Assessment of Cardiac Health and Thrombotic Risk in a Patient

ABSTRACT

The invention features methods and compositions for assessing risk, particularly immediate risk, of thrombotic events in patients with suspected or known vascular disease, and more particularly to assessing risk of thrombotic events in patients with coronary artery disease, particularly acute myocardial infarction, stroke, unstable angina, stable angina, or restenosis. Risk of thrombosis can be assessed by analysis of platelet reactivity and/or velocity of thrombin or fibrin formation, and determining whether the patient has a score associated above a risk threshold value. In other embodiments, risk of thrombosis in a patient is evaluated in the context of a profile generated from values obtained from one or more assays that evaluate various factors associated with thrombosis and/or atherosclerosis.

FIELD OF THE INVENTION

The present invention relates generally to assessing risk of athrombotic event and associated cardiac events, including assessing riskof ischemic and bleeding events, particularly acute myocardialinfarction, in patients with suspected or known vascular disease.

BACKGROUND OF THE INVENTION

Preventative care and selection of therapy for patients with suspectedor known vascular disease remains a difficult task for clinicians. Thereare no currently available methods that completely assess the immediaterisk of developing myocardial ischemia or infarction from thrombosis oratherosclerosis. Importantly, there are significant limitations of themethods now used to assess the acute risk of thrombosis or bleeding inpatients with suspected or known vascular disease, particularly wherethe patient is receiving anti-thrombotic therapy. In addition,percutaneous intervention can increase the risk of a thrombotic eventand therapies used to decrease this risk can cause bleeding.

At present, available indicators are only indirect methods for assessingsuch risks, since they do not reflect the risk of the final event invessel occlusion and thrombosis. Indirect measurements include:

-   -   1. Cholesterol level: High cholesterol levels are associated        with the formation of atherosclerotic plaque formation;    -   2. LDL cholesterol level: This “bad” type of cholesterol, with        high levels associated with accelerated atherosclerotic plaque        formation;    -   3. LDL cholesterol particle size: Particle size is another        measurement of the “bad” type of cholesterol, often signifying a        genetic predisposition towards the development of        atherosclerosis. Small LDL particles can be seen in various        hyperlipidemic states and the metabolic syndrome;    -   4. HDL cholesterol level: This “good” type of cholesterol, with        high levels being a positive predictor in controlling the        development of atherosclerosis;    -   5. HDL cholesterol particle size: Particle size is another        measurement of this “good” type of cholesterol with large        particles conferring the most protection against        atherosclerosis;    -   6. Triglyceride level: High levels are associated with various        hyperlipidemic states and the metabolic syndrome which        predispose patients to atherosclerosis;    -   7. Glucose measurements: Glucose intolerance and diabetes        mellitus are associated with accelerated atherosclerosis        (coronary heart disease and peripheral vascular disease).        Glucose levels can be measured by standard assays. The        hemoglobin A1C (glycosylated hemoglobin) is a measure of overall        glycemic control. The glucose tolerance test indicates a        patient's ability to clear glucose from the circulation        following a glucose challenge;    -   8. Homocysteine levels: High levels are associated with        accelerated atherosclerotic plaque formation;    -   9. Inflammatory markers: High levels of markers such as, e.g.,        C-reactive protein, Interleukin-6, and myeloperoxidase, are        associated with acute myocardial infarction and the development        of atherosclerosis;    -   10. Assessment of coagulation status: Coagulation status, as        assessed by, for example, prothrombin time (PT), International        Normalized Ratio (INR) and partial thromboplastin time (PTT) are        insufficient measures of thrombosis risk and are used to gauge        anti-coagulation status (blood-thinning);

Assessing risk of a thrombotic event is of particular importance tomedical treatment of all patients where coagulation is concerned. Forinstance, when a patient with vascular disease is about to undergo orhas undergone percutaneous intervention to relieve an arterial orvascular stenosis. Such interventions include angioplasty and stenting.Such patients are routinely treated with a variety of thrombosisinhibitors (e.g., anti-platelet drugs) to reduce the risk of acutevessel occlusion. However, at the same time, such inhibitors increasethe risk of bleeding. Despite the risks, such thrombosis inhibitors aregenerally administered in the same dose to all patients. Since there areno universally accepted methods to assess the risk of thrombosis andacute myocardial infarction in a patient, clinicians choose instead totreat the patient with a standard dose of drags in hopes of avoiding anevent having an unknown risk. As a result, it is likely that manypatients are unnecessarily over-treated with drug(s) that are associatedwith dangerous, life-threatening side effects. Alternatively, somepatients do not achieve adequate protection against thrombosis.

There is a need in the field for improved or alternative methods forassessing risk of thrombosis and immediate risk of a cardiac event in apatient, as well as methods for tailoring therapy based on this riskand/or the responsiveness of a patient to therapy. The present inventionaddresses these needs.

LITERATURE

-   Matetzky et al. Circulation. 2004; 109:3171-5; Kabbani et al. Am J    Cardiol. 2003; 91:876-8; Kabbani et al. Circulation. 2001;    104:181-6.-   Samara et al. Thromb Haemostat. 2005 115:89-94; Muller et al. Thromb    Haemost. 2003; 89:783-7; Gurbel et al. Circulation. 2003;    107:2908-13.-   Gurbel et al. Pharm Res 1999; 65: 109-23; Gurbel et al. Platelets.    2004; 15:95-9; Gurbel et al. Thromb Res 2003; 112:9-12; Barragan et    al. Catheter Cardiovasc Jnterv. 2003; 59:295-302; Gum et al. J Am    Coll Cardiol. 2003; 4:961-5; Regar et al. Am J Cardiol. 2004;    93:1271-5; Gurbel et al. Haematologica. 2004; 89; Supplement 7;    9-11; Zimmermann et al. Circulation. 2003; 108:542-7; Mobley et al.    Am J Cardiol. 2004; 93:456-8; Muller et al. Heart. 2001; 85:92-3;    Gurbel et al. Platelets. 2003; 14:481-3; Gurbel et al. J Thromb    Haemost. 2003; 1:1319-21; Gurbel et al. Expert Rev Cardiovasc Ther.    2004; 2:535-45

SUMMARY OF THE INVENTION

The invention features methods and compositions for assessing risk,particularly the immediate risk, of a thrombotic event in a patient,including myocardial ischemia from thrombotic and bleeding events, acutemyocardial infarction, stroke, unstable angina, stable angina, orrestenosis. Assessment in a patient with suspected or known vasculardisease is of particular interest.

In an embodiment of particular interest, risk of a thrombotic event (eg, immediate risk) is assessed by determining whether plateletreactivity score in a patient (e.g., by assessing platelet aggregationand other indicators associated with increased platelet reactivity) thatis above a risk threshold value. In another embodiment of particularinterest, risk of a thrombotic event (e.g., immediate risk) is assessedby determining whether a velocity of thrombin or fibrin formation score(e.g., by assessing time-to-thrombin formation or time-to-fibrinformation) is above a risk threshold value. In other embodiments,cardiac health, including the risk of a thrombotic event, in a patientis evaluated in the context of a profile generated from values obtainedfrom one or more assays that evaluate various factors associated with,for example, thrombosis and/or atherosclerosis.

In one aspect the invention features a method of assessing risk of athrombotic event in a patient by assessing platelet reactivity,time-to-thrombin formation (TTF), or time-to-fibrin formation (TFF) in ablood sample of a patient having or suspected of having vasculardisease, said assessing providing a test score, wherein a test scoregreater than a risk threshold score indicates the patient is at risk ofa thrombotic event.

In a related embodiment, the thrombotic event is a thrombosis,particularly a stent thrombosis. In further related embodiments,platelet reactivity is assessed independent of a pre-treatment baselineof platelet reactivity in the patient. In specific relatedembodiments, 1) platelet reactivity is assessed by 5 μM ADP-inducedplatelet aggregation and the risk threshold score is from about 24% to36%; 2) platelet reactivity is assessed by 20 μM ADP-induced plateletaggregation and the risk threshold score is from about 40% to 60%; 3)platelet reactivity is assessed by a P2Y₁₂ reactivity ratio and the riskthreshold score is from about 32 to about 48; and/or 4) plateletreactivity is assessed by stimulated GP expression and the riskthreshold score is from about 32 to about 48. In one embodiment,platelet reactivity is assessed by a method other thanthromboelastography maximum amplitude (MA).

In another embodiment, the methods are used to assess whether the riskof a thrombotic event is an immediate risk of a thrombotic event. Inrelated embodiments, the immediate risk of a thrombotic event is risk ofa thrombotic event within about 18 months, or within about 6 months. Infurther related embodiments, thrombotic event is a recurrent thromboticevent, such as myocardial ischemia.

In a related embodiment immediate risk of a thrombotic event is assessedby assessing at least one of platelet reactivity, time-to-thrombinformation (TTF) or time-to-fibrin formation (TFF), and further where 1)platelet reactivity is assessed by thromboelastography Maximum Amplitude(MA), the risk threshold score is from about 58 to 86; 2) plateletreactivity is assessed by 5 μM ADP-induced platelet aggregation, therisk threshold score is from about 45% to 55%; 3) platelet reactivity isassessed by 20 μM ADP-induced platelet aggregation, the risk thresholdscore is from about 52% to 76%; 4) where when TTF and TFF are assessedby thromboelastography R and the risk threshold score is from about 4.6min to 5.6 min.

In further related embodiments, the thrombotic event is myocardialischemia, myocardial infarction, unstable angina, stable angina,restenosis, stroke or deep vein thrombosis. In further embodiments, riskis assessed prior to percutaneous intervention or pharmacologicalintervention and/or in a patient undergoing therapy, e.g., with ananti-platelet inhibitor.

In still other embodiments, the methods further include modifyingtherapy to modulate a risk factor for a thrombotic event, e.g., toreduce platelet reactivity, or to increase at least one of TTF and TFF,in the patient.

In another aspect the invention features methods of assessing cardiachealth in a patient by assessing at least one of platelet reactivity,time-to-thrombin formation (TTF), or time-to-fibrin formation (TFF) in ablood sample of a patient in a blood sample of a patient, said assessingproviding a test score, where comparison of the test score to a riskthreshold score is indicative of cardiac health in the patient.

In a related embodiment, platelet reactivity is assessed, and 1) whereplatelet reactivity is assessed by 5 μM ADP-induced plateletaggregation, a risk threshold score of from about 24% to 36% isindicative of risk of a thrombotic event in the patient; 2) whereplatelet reactivity is assessed by 20 μM ADP-induced plateletaggregation, a risk threshold score of from about 40% to 60% isindicative of risk of a thrombotic event in the patient; 3) whereplatelet reactivity is assessed by a P2Y₁₂ reactivity ratio, a riskthreshold score of from about 32 to about 48 is indicative of risk of athrombotic event in the patient; or 4) where platelet reactivity isassessed by stimulated GP IIb/IIIa expression, a risk threshold score offrom about 32 to about 48 is indicative of risk of a thrombotic event inthe patient.

In further related embodiments, platelet reactivity is assessed, andfurther 1) where platelet reactivity is assessed by thromboelastographyMaximum Amplitude (MA), a risk threshold score of from about 58 to 86 isindicative of immediate risk of a thrombotic event in the patient; 2)where platelet reactivity is assessed by 5 μM ADP-induced plateletaggregation, a risk threshold score of from about 45% to 55% isindicative of immediate risk of a thrombotic event in the patient;and/or 3) where platelet reactivity is assessed by 20 μM ADP-inducedplatelet aggregation, a risk threshold score of from about 52% to 76% isindicative of immediate risk of a thrombotic event in the patient.

In another related embodiment, at least one of TTF or TFF is assessed.Where TTF and TFF are assessed by thromboelastography R, a riskthreshold score of from about 4.6 min to 5.6 min is indicative ofimmediate risk of a thrombotic event in the patient.

In still other related embodiments, the method of assessing cardiachealth involves assessing one or more of a thrombotic event risk factorselected from a lipid risk factor, an inflammation risk factor, aoxidation marker, or a metabolic risk factor. The lipid risk factor istotal cholesterol level, LDL cholesterol level, LDL cholesterol particlesize, HDL cholesterol level, HDL cholesterol particle size, triglyceridelevel, LPa, or Lp-PLA2; the method of claim 26, wherein the inflammationrisk factor is a level of C-reactive protein, a level of IL-6, or alevel of ICAM-1; the oxidation marker is a level of myeloperoxidase, alevel of oxidized LDL, or a level of an oxidized fatty acid; and themetabolic risk factor is fasting glucose, a level of hemoglobin A1C, ora homocysteine level.

In another aspect the invention features methods of assessing risk ofstent thrombosis in a patient, the method comprising assessing plateletreactivity in a blood sample of a patient having or suspected of havingvascular disease, where said assessing provides a platelet reactivitytest score, wherein a platelet reactivity test score greater than a riskthreshold score indicates the patient is at risk of stent thrombosis. Inrelated embodiments, platelet reactivity is assessed independent of apre-treatment baseline of platelet reactivity in the patient.

In one embodiment, platelet reactivity is assessed 1) by 5 μMADP-induced platelet aggregation, and the risk threshold score is fromabout 24% to 36%; 2) by 20 μM ADP-induced platelet aggregation, and therisk threshold score is from about 40% to 60%; 3) by P2Y₁₂ reactivityratio, and the risk threshold score is from about 32 to about 48; and/or4) by a level f stimulated GP IIb/IIIa expression, the risk thresholdscore is from about 32 to about 48. Preferably platelet reactivity isassessed by a method other than thromboelastography maximum amplitude(MA). In further related embodiments, assessment is performed is priorto percutaneous intervention or pharmacological intervention, and/or ina patient is undergoing therapy with an anti-platelet inhibitor (e.g.,an ADP-induced platelet aggregation inhibitor). In further embodiment,the method further includes modifying therapy to reduce plateletaggregation in the patient.

In another aspect the invention features a method of assessing immediaterisk of a thrombotic event in a patient, the method comprising assessingplatelet reactivity, time-to-thrombin formation (TTF), or time-to-fibrinformation (TFF) in a blood sample of a patient having or suspected ofhaving vascular disease, said assessing providing a test score; wherecomparison of the test score greater to a immediate risk threshold valueis indicative of immediate risk of myocardial ischemia in the patient.

In a related embodiment, platelet reactivity is assessed, and 1) whenplatelet reactivity is assessed by thromboelastography Maximum Amplitude(MA), the risk threshold score is from about 58 to 86; 2) when plateletreactivity is assessed by 5 μM ADP-induced platelet aggregation, therisk threshold score is from about 45% to 55%; and 3) when plateletreactivity is assessed by 20 μM ADP-induced platelet aggregation and therisk threshold score is from about 52% to 76%. In a further relatedembodiment, at least one of TTF or TFF is assessed, and further wherewhen TTF and TFF are assessed by thromboelastography R and the riskthreshold score is from about 4.6 min to 5.6 min.

In another aspect, the invention features a method of treating avascular disease in a patient, the method comprising administering atreatment regimen comprising administration of a active agent to apatient having vascular disease in an amount effective to decrease riskof a thrombotic event, wherein risk of the thrombotic event is assessedaccording to the methods described above and herein.

In another aspect the invention features a method of treating a vasculardisease in a patient, the method comprising administering a treatmentregimen comprising administration of a active agent to a patient havingvascular disease; assessing a risk of a thrombotic event in the patientaccording to the methods described above and herein; and adjusting thetreatment regimen so as to decrease the risk of a thrombotic event inthe patient.

The invention is advantageous in that, prior to the present invention,no readily available or accepted methodology was available to directlyassess risk of patients for a thrombotic event, including myocardialischemia or myocardial infarction. Platelets play a fundamental role inthe final event of vessel occlusion. Assessment of platelet reactivitypredicts which patients are at higher risk for complications. Thus theinvention provides the means to assess a patient's risk of differentthrombotic events such as heart attack, stroke, and deep venousthrombosis. In the peri-operative state for surgeries (vascular,orthopedic and abdominal surgeries), the invention can identify whichpatients are predisposed to acute thrombotic events. The type ofthrombotic event, for which the patient is at risk can readily bedetermined by the context in which the patient presents (e.g., in thecontext of other signs and symptoms associated with diagnosis of theunderlying condition or disease).

The invention provides the advantage that platelet aggregation providesa marker for risk of a thrombotic event independent of responsiveness todrug therapy (e.g., as assessed by a change in platelet reactivityfollowing administration of an anti-platelet drug such as clopidogrel,often referred to “drug responsiveness”, e.g., “clopidogrelresponsiveness”). For example, the inventor has found that clopidogrelresistance may overestimate the risk of stent thrombosis innon-responders with low pre-treatment platelet aggregation, andunderestimate the risk of stent thrombosis in responders with highpost-treatment platelet aggregation. Further, there is no need to assessplatelet reactivity relative to a baseline of reactivity prior toadministration of a therapy. Instead, a “raw” platelet reactivity scorecan be used to assess risk of for example, stent thrombosis ormyocardial ischemia (such as myocardial infarction), and thus there isno need for a comparative analysis (e.g., between a pre-treatment scoreand a post-treatment score). As such the invention provides a method forassessing risk of a thrombotic event relative to a baseline of plateletreactivity, time-to-thrombin formation (TTF), or time-to-fibrinformation (TFF) that may have been present prior to drug-based therapyor other therapy.

The invention thus provides methods for assessing risk of a thromboticevent based on intrinsic platelet reactivity, TTF or RFF valuesindependent of the baseline, and regardless of the presence or absenceof the effect of any drugs or other therapy upon these characteristics.

The invention also provides methods and compositions that provide forcoordination of results of various tests for risk factors associatedwith a thrombotic event (e.g., myocardial ischemia, atherosclerosis).

These and other advantages will be apparent to the ordinarily skilledartisan upon reviewing the present specification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing ADP-induced platelet aggregation (5 μM) inpatients with stent thrombosis (SAT) and without stent thrombosis (NoSAT).

FIG. 2 is a graph showing ADP-induced Platelet aggregation (20 μM) inpatients with stent thrombosis (SAT) and without stent thrombosis (NoSAT).

FIG. 3 is a graph showing thromboelastography MA analysis in patientswith stent thrombosis (SAT) and without stent thrombosis (No SAT).

FIG. 4 is a graph showing platelet inhibition in response to 5 μM ADPfollowing the 4 treatment regimens. Group A=300 mg clopidogrel; GroupB=600 mg clopidogrel; Group C=300 mg clopidogrel+eptifibatide; and GroupD=300 mg clopidogrel+eptifibatide. *p≤0.001, Group A vs. B; o p<0.001,Groups C or D vs. Groups A or B.

FIG. 5 is a graph showing platelet inhibition in response to 20 μM ADPfollowing the 4 treatment regimens. Group A=300 mg clopidogrel; GroupB=600 mg clopidogrel; Group C=300 mg clopidogrel+eptifibatide; and GroupD=300 mg clopidogrel+eptifibatide. *p=0.001, Group A vs. B.+p=0.01,Group A vs. B. o p<0.001, Groups C and D vs. Groups A and B δ p=0.05,Group C vs. D.

FIG. 6 is a graph showing stimulated p-selectin expression at baselineand 18-24 hours post-stenting. *p≤0.02 vs. baseline; +p≤0.03 vs. GroupsB, C, or D. Group A=300 mg clopidogrel; Group B=600 mg clopidogrel;Group C=300 mg clopidogrel+eptifibatide; and Group D=300 mg clopidogreleptifibatide.

FIG. 7 is a graph showing relative inhibition of active GP IIb/IIIaexpression at 18-24 hours post-stenting. Group A=300 mg clopidogrel;Group B=600 mg clopidogrel; Group C=300 mg clopidogrel+eptifibatide; andGroup D=300 mg clopidogrel+eptifibatide.

FIG. 8 is a bar graph demonstrating CK-MB release in the 4 treatmentgroups. * p<0.05 between groups C and D vs. A and B. Group A=300 mgclopidogrel; Group B=600 mg clopidogrel; Group C=300 mgclopidogrel+eptifibatide; and Group D=300 mg clopidogrel+eptifibatide.ULN=upper limit of normal value.

FIG. 9 is a bar graph demonstrating myoglobin and troponin release inthe 4 treatment groups. Group A=300 mg clopidogrel; Group B=600 mgclopidogrel; Group C=300 mg clopidogrel+eptifibatide; and Group D=300 mgclopidogrel+eptifibatide. ULN=upper limit of normal value.

FIG. 10 is a bar graph demonstrating mean platelet reactivity measuredover 18-24 hours post-stenting by 5 μM ADP-induced aggregation. +p=0.002between groups A and B, *p<0.001 between either Group C or D vs. Group Aor B. Group A=300 mg clopidogrel; Group B=600 mg clopidogrel; GroupC=300 mg clopidogrel+eptifibatide; and Group D=300 mgclopidogrel+eptifibatide.

FIG. 11 is a graph showing the relation of necrosis marker release(CKMB) to mean platelet reactivity as measured by 5 μM ADP-inducedaggregation. NL=normal limit; ULN=upper limit of normal value.

FIG. 12 is a graph showing the relation of myocardial infarction to meanplatelet reactivity as measured by 20 μM ADP-induced aggregation.

FIG. 13 is a graph demonstrating onset of first ischemic event.

FIG. 14 is ADP induced post-treatment platelet aggregation (20 uM ADP)measured by light transmittance aggregometry (LTA) in patients withoutischemic events and with ischemic events.

FIG. 15 is a distribution of patients with events into quartiles and theincidence of events in each quartile as measured by light transmittanceaggregometry (20 uM ADP). P values are in comparison with highestquartile.

FIG. 16 is clot strength (MA) measured by thromboelastography inpatients without ischemic events and with ischemic events.

FIG. 17 is distribution of patients with events into quartiles and theincidence of events in each quartile as measured by maximum amplitude(thromboelastography MA) P values are in comparison with highestquartile.

FIG. 18 is reaction time R measured by thromboelastography in patientswith and without ischemic events.

FIG. 19 is distribution of patients with events into quartiles and theincidence of events in each quartile as measured by Reaction Time(thromboelastography R) P values are in comparison with lowest quartile.

FIG. 20 is a flowchart exemplifying the computer program of theinvention.

DEFINITIONS

A “threshold score” or “risk threshold score” is a value for an assayfor biological assay, e.g., platelet reactivity, time-to-thrombinformation (TTF), or time-to-fibrin formation (TFF), which is anapproximate level, point, or value which distinguishes a relatively highrisk of an event from a relatively low risk of an event. For example, inthe context of platelet reactivity, in most cases the threshold score isan approximate value above which risk of a thrombotic event isrelatively higher and below which risk of a thrombotic event isrelatively lower. In the context of TTF and TFF, in most cases thethreshold score is an approximate value below which risk of a thromboticevent is relatively higher and above which risk of a thrombotic event isrelatively lower.

As used herein “borderline” refers to a platelet reactivity score thatis about 10% above or below a risk threshold value, and “abnormal” isgreater than or at a risk threshold value. Where a value above a riskthreshold value indicates an increased risk (or immediate risk) of athrombotic event, a patient can “borderline abnormal” if above the riskthreshold value, but at less than or about 10% above the risk thresholdvalue; likewise, a patient can be “borderline normal” if below the riskthreshold value, but at less than or about 10% below the threshold.Similarly, where a value below a risk threshold value indicates anincreased risk (or immediate risk) of a thrombotic event, a patient can“borderline abnormal” if below the threshold value, but at less than orabout 10% below the risk threshold value; likewise, a patient can be“borderline normal” if above the platelet reactivity risk thresholdvalue, but at less than or about 10% above the threshold.

A “platelet reactivity score” as used herein is meant to refer to avalue obtained from assessment of platelet reactivity in a patient,where platelet reactivity is assessed by, for example conventional lighttransmittance aggregometry (for example, ADP-induced plateletaggregation) (e.g., 5 μM or 20 μM ADP) or thromboelastography.

A “time-to-thrombin formation (TTF) score” or “time-to-fibrin formation(TFF) score” is meant to refer to a value (usually expressed in minutesor seconds) obtained from assessment of TTF or TFF (e.g., bythromboelastography).

A “thrombotic event’ is meant to include events associated with anarterial or vascular thrombus such as myocardial ischemia, heart attack(myocardial infarction, including acute myocardial infarction), stroke,cardiovascular death, angina (e.g., unstable angina), stent thrombosis,(including thrombosis associated with percutaneous intervention (e.g.,stent thrombosis, stent re-stenosis) or other surgeries (e.g., vascular,orthopedic, or abdominal surgeries), deep venous thrombosis, stentre-stenosis, pulmonary embolus, and the like, particularly those whichcan lead to serious morbidity and/or mortality.

“Myocardial ischemia” as used herein refers to a low oxygen state due toobstruction of the arterial blood supply or inadequate blood flowleading to hypoxia in myocardial tissue. Thrombosis and atherosclerosisare two causes of myocardial ischemia referred to herein.

“Thrombosis” as used herein refers to the presence of a thrombus, anaggregation of blood factors, primarily platelets and fibrin withentrapment of cellular elements, which causes vascular obstruction atthe point of formation.

“Atherosclerosis” as used herein refers to the progressive narrowing andhardening of the arteries over time so as to obstruct blood flow. Thisprocess generally occurs to some degree with age, but is acceleratedwith risk factors such as high cholesterol, high blood pressure,smoking, diabetes, and family history for atherosclerotic disease.

“Stenosis” as used herein refers to a narrowing of a vessel witharterial stenosis or vascular stenosis being of particular relevance tothe present invention.

“Restenosis” as used herein refers to recurrence of stenosis aftercorrective surgery to remove or reduce a previous narrowing of anartery. Restenosis occurs most commonly after percutaneous interventionto treat atherosclerotic narrowings secondary to plaque buildup andthrombosis in arteries.

The term “biological sample” encompasses a variety of sample typesobtained from an organism and can be used in a diagnostic or monitoringassay. The term encompasses blood and other liquid samples of biologicalorigin, solid tissue samples, such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. The termencompasses samples that have been manipulated in any way after theirprocurement, such as by treatment with reagents, solubilization, orenrichment for certain components. The term encompasses a clinicalsample, and also includes cells in cell culture, cell supernatants, celllysates, serum, plasma, biological fluids, and tissue samples.

The terms “body fluid” and “bodily fluid,” used interchangeably herein,refer to a biological sample of liquid from a mammal, e.g., from ahuman. Such fluids include aqueous fluids such as serum, plasma, lymphfluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid,milk, whole blood, urine, cerebrospinal fluid, saliva, sputum, tears,perspiration, mucus, tissue culture medium, tissue extracts, andcellular extracts. Particular bodily fluids that are interest in thecontext of the present invention include whole blood, serum, plasma, andother blood-derived samples, wherein the term “blood sample” is meant toencompass all such samples, with appropriate types of samples beingselected according to any assay to be conducted.

A “computer-based system” refers to the hardware means, software means,and data storage means used to analyze information. The minimum hardwareof a patient computer-based system comprises a central processing unit(CPU), input means, output means, and data storage means. A skilledartisan can readily appreciate that any one of the currently availablecomputer-based system are suitable for use in the present invention. Thedata storage means may comprise any manufacture comprising a recordingof the present information as described above, or a memory access meansthat can access such a manufacture.

To “record” data, programming or other information on a computerreadable medium refers to a process for storing information, using anysuch methods as known in the art. Any convenient data storage structuremay be chosen, based on the means used to access the stored information.A variety of data processor programs and formats can be used forstorage, e.g. word processing text file, database format, etc.

A “processor” or “computing means” references any hardware and/orsoftware combination that will perform the functions required of it. Forexample, any processor herein may be a programmable digitalmicroprocessor such as available in the form of a electronic controller,mainframe, server or personal computer (desktop or portable). Where theprocessor is programmable, suitable programming can be communicated froma remote location to the processor, or previously saved in a computerprogram product (such as a portable or fixed computer readable storagemedium, whether magnetic, optical or solid state device based). Forexample, a magnetic medium or optical disk may carry the programming,and can be read by a suitable reader communicating with each processorat its corresponding station.

By “clinical assay” is meant an assay or test that is performed on asample obtained from an individual or patient (also referred to hereinas host or patient) in order to provide information on current or futurehealth or condition, diagnosis, treatment, prevention, and/or monitoringof a condition of the individual or patient.

The terms “evaluate” and “assess” are used interchangeably hereinbroadly to refer not only to the diagnosis or detection of a givencondition of interest, but also to the monitoring of a condition over agiven period of time. As such, in certain embodiments one uses thepatient methods to assess the efficacy of a given treatment regimen fora given condition (e.g., a thrombotic or atherosclerotic condition.). Inyet other embodiments, one uses the patient methods to monitor, predict,or track (i.e., watch or observe), the progression of a condition in apatient over a period of time.

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment,” as used herein, covers anytreatment of a disease in a mammal, particularly in a human, and caninclude: (a) preventing the disease or a symptom of a disease fromoccurring in a patient which may be predisposed to the disease but hasnot yet been diagnosed as having it (e.g., including diseases that maybe associated with or caused by a primary disease); (b) inhibiting thedisease or condition, i.e., arresting its development; and (c) relievingthe disease, i.e., causing regression of the disease.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, patientto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “asample” includes a plurality of such samples and reference to “the drug”includes reference to one or drugs and equivalents thereof known tothose skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that plateletreactivity, and more particularly platelet aggregation is a marker ofthe risk and occurrence of thrombotic events, including myocardialischemic events in a patient having or suspected of having vasculardisease, particularly in patients who have undergone percutaneousintervention and may be at acute risk of, for example, stent thrombosis,vessel restenosis, myocardial infarction and stroke. Importantly,platelet reactivity (e.g., as assessed by platelet reactivity), time tothrombin generation (also referred to herein as time-to-thrombinformation (TTF)), and time-to-fibrin generation (also referred to hereinas time-to-fibrin formation (TFF)), are effective markers for risk of athrombotic event, such as myocardial ischemia, independent ofresponsiveness to drug therapy (e.g., as assessed by a change inplatelet reactivity following administration of an anti-platelet drugsuch as clopidogrel, often referred to “drug responsiveness”, e.g.,“clopidogrel responsiveness”).

Prior to the present invention, there was no readily available oraccepted methodology to assess these patients; the invention providessuch a methodology. More importantly, knowledge of a patient's plateletreactivity, TTF and TFF are invaluable in preventing complicationsrelated to surgical and percutaneous vascular procedures (e.g., stentplacement or balloon angioplasty) such as stent thrombosis orre-stenosis. Furthermore, assessing risk of patients to thromboticevents provides a means to facilitate selection of therapy (e.g., dose,regimen, anti-platelet therapy, and the like) so as to better predicttherapeutic efficacy.

The invention is based on the discovery that platelet reactivity, TTFand TFF provide a powerful and direct predictor of risk of seriouscomplications in patients with vascular disease, such as acutethrombosis and myocardial infarction. In particular, the inventor hasdiscovered that platelet reactivity, TTF and TFF directly predicts theoccurrence of thrombosis in patients. For instance, platelet reactivitymeasured by ADP-induced aggregation and thrombin generation, as measuredby thromboelastography (MA) directly correlates with the risk andoccurrence of ischemic coronary events and coronary artery stentthrombosis.

The invention also addresses the need to provide a means to compile andcorrelate data from various assays that test various thrombosis and/oratherosclerosis risk factors. For example, the results of a plateletaggregation assay and assessment of risk as provided by the inventioncan be provided along with the results of various other assays so as toprovide a patient's profile of risk for a thrombotic event (e.g.,thrombosis and/or atherosclerosis). These methods can be implemented bya computer program which can be internet based, and which are designedto facilitate the use of the test results by medical professionals tohave an enhanced understanding of the patient's individual profile andthe most appropriate care to be administered.

The invention will now be described in more detail.

Thrombotic Events, Including Myocardial Ischemia

The methods and compositions of the invention can be used to assess riskof any of a variety of thrombotic events, including myocardial ischemicsyndromes, including those associated with thrombosis and withatherosclerosis. Thrombotic events includes events such as heart attack(myocardial infarction, particularly acute myocardial infarction),stroke, cardiovascular death, angina, stent thrombosis, stentre-stenosis, deep venous thrombosis, pulmonary embolus, and the like.

The invention is the most immediate and direct assessment of a patient'srisk for thrombosis (particularly stent thrombosis) and adverse ischemicevents by measuring platelet reactivity, e.g., as assessed by plateletaggregation induced by ADP and thrombin, and/or TTF and/or TFF.Assessment of risk of thrombosis is of particular importance indetermining a patient's risk prior to percutaneous therapy, particularlypercutaneous therapy involving implantation of a stent. Thrombosis inthis context can lead to a serious event that can and often does resultin death during and after the procedure.

The methods of the invention find particular use in diagnosis and riskassessment of a variety of thrombotic or atherosclerotic events. Suchthrombotic events include, but are not necessarily limited to myocardialinfarction, particularly acute myocardial infarction. Acute myocardialinfarction is death of myocardial tissue that is acute and secondary toocclusion of an artery that provides blood flow to the affected tissue.This can occur at any time.

Assessing Risk of a Thrombotic Event

The invention features methods for assessing a patient's risk of athrombotic event (e.g., myocardial ischemia) by assessing plateletreactivity, and may also be assessed by assessing time-to-thrombinformation (TTF), and/or time-to-fibrin formation (TFF). The inventionalso features methods for assessing a patient's immediate risk of athrombotic event (e.g., a thrombotic event such as myocardial ischemia,particularly a recurrent myocardial ischemia within about 6 months byassessing platelet reactivity, time-to-thrombin formation (TTF), and/ortime-to-fibrin formation (TFF).

Commercially available devices and assays can be used to measureplatelet reactivity, TTF, and TFF. These devices include lighttransmittance aggregometers (LTA); thromboelastography instruments(e.g., MA, R); single platelet counters (Plateletworks); flow cytometryto measure activation of platelets, microparticles andplatelet-procoagulant activity (phosphatidyl-serine expression); methodsto measure thrombosis after application of shear stress to a bloodsample (Dade-Behring PFA-100); and methods to assess inhibition ofplatelets by specific drugs (aspirin and clopidogrel (Accumetrics)).Assessment of platelet reactivity, TTF and TFF can thus generally beaccomplished in several different ways according to the invention.

In one embodiment, the method of the invention involves assessment ofplatelet reactivity to provide for a platelet reactivity score. Thisplatelet reactivity score is analyzed to determine whether the plateletreactivity score is above, below or at a threshold value, where thethreshold value demarcates a level of risk of a cardiac event,particularly a thrombotic event (e.g., stent thrombosis, myocardialischemia, etc.). If a patient has a platelet reactivity score above arisk threshold value, then the patient is at an increased risk of, forexample, stent thrombosis or myocardial. If a patient has a plateletreactivity score below a risk threshold value, then the patient has adecreased risk of, for example, stent thrombosis or myocardial ischemia.If a patient has a borderline or abnormal platelet reactivity score,then close monitoring of the patient is warranted.

Platelet reactivity can be assessed by any appropriate assay thatassesses a change in function (e.g., induction of platelet aggregation),intracellular signaling pathways (e.g., VASP phosphorylation), orsurface receptor expression (e.g., GP IIa/IIIb expression; p-selectin;PCAM-I, CD40 ligand, and the like) in the presence of a stimulusassociated with clot formation. Methods for assessing plateletreactivity in terms of a change in function, intracellular signalingpathways, or surface receptor expression are well known the art.

For example, in one embodiment, platelet reactivity is assessed throughan ADP-induced platelet aggregation assay. Methods for conducting suchassays are well known in the art, and examples of the use of such assaysare detailed in the Examples section below.

In general, the platelet aggregation assay is conducted to provide aplatelet aggregation score. As used herein, a “platelet aggregationscore” (“PA score”), is defined as a measure of the reactivity ofplatelets to various stimuli. Because the PA score value variesaccording to the conditions under which the assay is conducted, “PA-Xn”refers to a PA score determined in the presence of a stimulant “X”,where “n” represents the concentration of the stimulant, where relevant(e.g., ADP at concentration of “n” μM). For example, “PA-ADP5” refers toa PA score determined in the presence of 5 μM ADP, while PA-ADP20 refersto a PA score determined in the presence of 20 μM ADP.

A patient's PA score is compared to a risk threshold PA score. If thepatient PA score is greater than a risk threshold PA score, then thepatient is at an increased risk of a thrombotic event. If the PA scoreis lower than a risk threshold PA score, then the patient has a lowerrisk of a thrombotic event. Because the platelet aggregation assay canbe sensitive to the concentration of ADP used to induce aggregation, therisk threshold differs according to the ADP concentration used in theassay.

In another exemplary embodiment, platelet reactivity is assessed bythromboelastography Maximum Amplitude (MA), which reflects strength of aclot, which in turn is dependent on number and function of platelets andits interaction with fibrin. In general, the thromboelastography is anavailable viscoelastic tests that characterizes formation and strengthof the blood clot over time, and can be used to measure in vitro and exvivo the life of a clot, the time to initial clot formation (time tothrombin generation or time to fibrin generation), then evaluate theacceleration phase of the developing clot, as well as its strengtheningand retraction. Thromboelastography can also be used to detect clotlysis.

Methods for assessing platelet reactivity by thromboelastography areknown in the art. In general, a sample of activated whole blood isplaced into a container such as a cuvette, and a suspended pistonlowered into the cuvette and is moved in rotation of a 4.5 degree arcbackwards and forwards. The fiber strands which interact with activatedplatelets attach to the surface of the cuvette and the suspended piston.The clot forming in the cuvette transmits its movement onto thesuspended piston. A “weak” clot stretches and therefore delays the arcmovement of the piston, which is graphically expressed as a narrowthromboelastography. A strong clot in contrary will move the pistonsimultaneously and proportionally to the cuvettes movements, creating athick thromboelastography. There are five parameters of thethromboelastography tracing: R, k, alpha angle, MA and MA60, whichmeasure different stages of clot development. R (reaction time) is aperiod of time from initiation of the test to the initial fibrinformation; k is measure of time from beginning of clot formation untilthe amplitude of thromboelastography reaches 20 mm, and represents thedynamics of clot formation. The alpha angle is an angle between the linein the middle of the thromboelastography tracing and the line tangentialto the developing “body” of the thromboelastography tracing. The alphaangle represents the acceleration (kinetics) of fibrin build up andcross-linking. MA is the Maximum Amplitude and reflects strength of aclot, which in turn is dependent on number and function of platelets andits interaction with fibrin. The MA60 is a measure of the rate ofamplitude reduction 60 min. after MA and represents the stability of theclot. In general, as used herein thromboelastography values are providedas MA values.

Additional examples of assessing platelet reactivity are provided in theExamples section below. In general, platelet reactivity can be assessedby any appropriate assay that assesses a change in function (e.g.,induction of platelet aggregation), intracellular signaling pathways(e.g., VASP phosphorylation), or surface receptor expression (e.g., GPIIa/IIIb expression; p-selectin; PCAM-I, CD40 ligand, and the like) inthe presence of a stimulus associated with clot formation. Methods forassessing platelet reactivity in terms of a change in function,intracellular signaling pathways, or surface receptor expression arewell known the art.

Selection of the appropriate assay will generally depend upon thecircumstances for which risk of a thrombotic event is to be assessed,and will be readily apparent to the ordinarily skilled artisan uponreading of the present disclosure. For example, where the risk of athrombotic event such as a stent thrombosis is to be assessed, assessingplatelet reactivity by examining change in platelet function (e.g., byplatelet aggregation (e.g., in the presence of ADP), but preferably notby thromboelastography MA) is of particular interest. Where theimmediate risk of a thrombotic event (such as recurrent ischemia) is tobe assessed, assessing platelet reactivity (e.g., by plateletaggregation (e.g., in the presence of ADP) or by thromboelastography(e.g., thromboelastography MA)), by assessing TTF, or by assessing TFF(where thromboelastography R provides a measure of both TTF ofparticular interest.

Risk of Thrombotic Event

In one embodiment, the invention is based on the discovery thatoccurrence of adverse thrombotic events, particularly after percutaneousintervention (e.g., percutaneous coronary revascularization), is relatedto platelet reactivity (e.g., as measured by change in platelet function(e.g., platelet aggregation in the presence of ADP), change inintracellular signaling (e.g., VASP phosphorylation), or by change inplatelet receptor expression (e.g., expression of GP IIb/IIIa)), withthe proviso that platelet reactivity is assessed by a method other thanthromboelastography MA. For example as described herein, studies ofpatients who have suffered subacute thrombosis (SAT) show that meanplatelet reactivity is higher than in those patients who were free ofthis adverse event.

For example, in one embodiment, platelet reactivity as assessed byADP-induced platelet aggregation. In general, a threshold of about 30%or greater, platelet aggregation induced by 5 μM ADP (that is, a PA-ADP5score of about 30% or greater) defined about 80% of the patients withstent thrombosis (SAT) (FIG. 1). A threshold of about 42% or greater,platelet aggregation induced by 20 μM ADP (that is, a PA-ADP20 score ofabout 42% or greater) included about 100% of the patients with SAT,while and a threshold of about 50% or greater, platelet aggregationinduced by 20 μM ADP (a PA-ADP20 score of about 50% or greater) definedabout 80% of the patients with SAT (FIG. 2). Thus the risk threshold foraggregation to define about 80% of SAT events is a PA-ADP5 score ofabout 30% or greater or a PA-ADP20 score of about 50% or greater. Incontrast, measurement of platelet reactivity as assessed bythromboelastography MA was not as reliable a predictor of SAT (FIG. 3).Thus, where the risk of a patient for stent thrombosis is to be assessed(e.g., prior to surgery), platelet reactivity should be assessed usingan assay other than thromboelastography MA (e.g., platelet aggregationin the presence of ADP).

Specifically, Example 1 below illustrates that patients who had aplatelet aggregation (5 μM ADP) (PA-ADP5) score of 49±4% suffered a SATwhile patients who had a PA-ADP5 of 26±2% did not have SAT (p<0.001,n=120 pts). Patients who had a platelet aggregation (20 μM ADP)(PA-ADP20) score of 65±3% suffered SAT while patients who had a PA-ADP20of 46±2% did not (p<0.001, n=120 pts).

Example 2 below shows that the absence of infarcts in patientsundergoing elective PCI with lower than about 50% mean 5 μM ADP-inducedaggregation indicates a threshold effect. Thus a 5 μM ADP-inducedplatelet aggregation score of less than about 50%, regardless of thepercent reduction relative to pretreatment platelet aggregation, is atherapeutic target to which therapy should be tailored. (n=120 patients)

Thus a risk threshold value for platelet reactivity as assessed byPA-ADP5 is defined as being from about 24% to 36%, usually about 26% to34%, usually about 28% to 32%, usually about 30%, and can be about 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, or 36%, moreusually about 30%. A PA-ADP5 score above this PA-ADP5 risk thresholdvalue indicates an increases risk of a thrombotic event such as stentthrombosis in the patient.

A risk threshold value for platelet reactivity as assessed by PA-AD20 isdefined as being from about 40% to 60%, usually about 42% to 58%,usually about 46% to 56%, usually about 48% to 54%, usually about 40%,41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59% or 60%, more usually about 50%. A PA-ADP20 scoreabout this PA-ADP20 risk threshold score indicates an increased risk ofa thrombotic event such as stent thrombosis.

In another embodiment, risk of a thrombotic event (e.g., stentthrombosis) is assessed by a change in intracellular signaling, e.g., asassessed by VASP phosphorylation. As described in the Examples below, aP2Y₁₂ reactivity ratio can be calculated to assess VASP phosphorylation.A P2Y₁₂ reactivity ratio of 46±9 was associated with no SAT within 6months, while a P2Y₁₂ reactivity ratio of 69±5 was associated with SATwithin about 6 months (Table 3). Thus a risk threshold value for P2Y₁₂reactivity ratio is defined as a P2Y₁₂ reactivity ratio of from aboutThus, a P2Y₁₂ reactivity ratio score of from about 32 to about 48, fromabout 34 to 46, from about 36 to 44, from about 38 to 42, usually about32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48,more usually about 40. A P2Y₁₂ reactivity ratio score above this P2Y₁₂reactivity ratio risk threshold value indicates an increased risk of athrombotic event such as stent thrombosis in the patient.

In another embodiment, risk of a thrombotic event (e.g., stentthrombosis) is assessed by a change in receptor expression, e.g., asassessed by GP IIb/IIIa expression (e.g., as assessed by surfacereceptor density). As described in the Examples below, a stimulated GPIIb/IIIa expression level of 42±4 was associated with no SAT within 6months; a stimulated GP IIb/IIIa expression level of 138±19 wasassociated with SAT within 6 months (Table 3). Thus a risk thresholdvalue for stimulated GP IIb/IIIa expression is defined as a stimulatedGP IIb/IIIa expression of from about 32 to about 48, from about 34 to46, from about 36 to 44, from about 38 to 42, usually about 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, more usuallyabout 40. A stimulated GP IIb/IIIa expression score above thisstimulated GP IIb/IIIa expression risk threshold value indicates anincreased risk of a thrombotic event such as stent thrombosis in thepatient.

Assessment of Immediate Risk of a Thrombotic Event

In another aspect, the invention provides for assessment of immediaterisk of a thrombotic event, particularly an ischemic event, such asstroke, recurrent angina, and the like. In this aspect, risk can beassessed by examining platelet reactivity, TTF, or TFF. By “immediaterisk” as used herein is meant risk of a thrombotic event, particularly arecurrent thrombotic event (e.g., recurrent myocardial ischemia) withina period of months from the time of the assay (e.g., within about 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 months, usuallywithin 4 months to 12 months, more usually within 6 months to 12 months,still more usually within 6 months from the time of the assay). In someembodiments it may be desirable to assess immediate risk of a thromboticevent using one or more of such methods.

As discussed in the examples below in more detail, in patients whoseplatelet reactivity and TTF/TFF (as assessed by tbromboelastography R)was measured at discharge following percutaneous coronaryrevascularization. As discussed in the Examples below, plateletreactivity (e.g., as assessed by platelet aggregation andthromboelastography by the thromboelastography (MA)), as well as TTF andTFF (e.g., as assessed by thromboelastography R), are powerfulpredictors for the occurrence of adverse ischemic events within 6months.

For example, in a study of 192 patients about 63% of patients in thehighest MA quartile (at least about 72 thromboelastography (MA) score,usually greater than about 72 thromboelastography (MA) score) will havean ischemic event within 6 months of discharge as compared to about 8%in the second quartile, about 8% in the third quartile and about 2% infourth highest quartile (FIG. 17). Thus, a threshold defining thehighest two quartiles (MA of about 68 or greater) includes about 85% ofall patients having an ischemic event within a 6 month period. Thus athromboelastography MA score of about 58 to 86, usually about 60 to 84,usually about 62 to 82, usually about 64 to 80, more usually about 66 to78, usually about 66 to 78, usually about 68 to 76, usually about 69 to74, usually about 71 to 73, more usually about 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, or 86, generally about 72 defines a risk threshold valuefor assessing immediate risk of a thrombotic event in a patient.

Platelet reactivity as assessed by ADP-induced platelet aggregation wasalso a reliable predictor of risk of an ischemic event within about 6months. Of the patients examined in the studies described herein, about35% of patients in the highest aggregation (20 μM ADP) quartile (greaterthan about 67% PA-ADP20) will have an ischemic event within 6 months ofdischarge as compared to 20%, 24%, and 10% in the second, third, andfourth highest quartiles, respectively (FIG. 15). Thus, using thethreshold defining the highest two quartiles (PA-ADP20 of about 61% orgreater) will include about 60% of all of those patients having anischemic event within about 6 months.

Finally, in patients whose platelet reactivity was measured seriallywhile in hospital following percutaneous revascularization (PCI), asillustrated herein development of myocardial infarction is dependent onplatelet reactivity. Using a threshold of about 50% 5 μM ADP-inducedaggregation included all of those patients who suffered a myocardialinfarction (CKMB>3× upper limits normal) (FIG. 11). Using a threshold ofabout 64% 20 μM ADP-induced aggregation included all of those patientswho suffered a myocardial infarction (CKMB>3× upper limits normal) (FIG.12).

Thus, a PA-ADP5 score of about 45% to 55%, 47% to 53%, 49% to 51%,usually about 45% 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, or 55%,usually about 50% defines a risk threshold value for assessing immediaterisk of a thrombotic event particularly a recurrent thrombotic eventsuch as myocardial ischemia, in a patient.

Further, a PA-ADP20 score of from about 52% to 76%, usually about 54% to75%, usually from about 56% to 73%, usually from about 58% to 71%,usually from about 60% to 69%, usually about 62% to 67%, usually about52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, or 76%, more usuallyabout 64% defines a risk threshold value for assessing immediate risk ofa thrombotic event particularly a recurrent thrombotic event such asmyocardial ischemia, in a patient.

In another embodiment, assessment of immediate risk of a thromboticevent, particularly an ischemic event by examining TTF or TFF. Methodsfor assessing TTF and TFF are well known in the art. In one embodiment,thromboelastography R (a period of time from initiation of the test tothe initial fibrin formation) is used. Since fibrin formation isdependent upon thrombin formation, thromboelastography R can be used toapproximate both TTF and TFF. As described in the Examples in moredetail below, a thromboelastography R score of less than about 5.1minutes indicates the patient is at risk of a thrombotic event withinabout 6 months (FIG. 19). Thus a TFF score, and thus a TTF score, offrom about 4.6 min to 5.6 min, usually from about 4.8 min to 5.4 min,usually from about 4.9 min to 5.2 min, usually about 4.6 min, 4.7 min,4.8 min, 4.9 min, 5.0 min, 5.1 min, 5.2 min, 5.3 min, 5.4 min, 5.5 min,or 5.6 min, usually about 5.1 min defines a risk threshold value forassessing immediate risk of a thrombotic event, particularly a recurrentthrombotic event such as myocardial ischemia, in a patient.

Depending on the event to be assessed (e.g., SAT vs recurrent ischemiavs post-elective PCI myocardial infarction (MI)), based on the extensivecollection of data provided herein, applying thresholds of plateletaggregation by 5 μM and 20 μM ADP and thromboelastography can definehigh risk groups. The data summarized above and described in more detailbelow support the utility of both methodologies, specificallyconventional platelet aggregation to define high risk for SAT andperi-procedural MI, and the thromboelastography to define high risk forevents within 6 months of PCI.

Where platelet aggregation is assessed in the context of therapy, suchas an anti-platelet therapy (e.g., clopidogrel), platelet aggregation isassessed without regard to (i.e., independent of) a pretreatmentbaseline of platelet aggregation. The invention is based on thediscovery that high platelet reactivity and low TTF/TFF is associatedwith a high risk of an ischemic event, e.g., a thrombotic event,regardless of whether the values obtained reflect a patient who isresponsive or non-responsive to to therapy (e.g., regardless of whetherplatelet reactivity is reduced in response to administration of drug,e.g., an anti-platelet inhibitor such as clopidogrel).

When a patient's PA score is greater than the corresponding riskthreshold, then the patient is diagnosed as having a risk of thrombosis,which can result in acute myocardial infarction, myocardial ischemia,restenosis, or stroke.

Patients can be stratified into ranges based on inter-quartile levelsand stratified according to risk. In general, patients in or above the75th percentile for platelet aggregation have a high risk of subsequentadverse ischemic events within six months after coronary arterystenting. The data presented in the present specification indicate thatpatients within at least the 75^(th) percentile (the first quartile forADP-induced aggregation or by thromboelastography) are at about a 35-63%% risk of a thrombotic event within 6 months. Patients with ADP-inducedplatelet aggregation of at least about 50% by 5 μM ADP or at least about64% by 20 μM ADP post-percutaneous intervention (PCI) are at risk formyocardial infarction (MI) in-hospital. Below these thresholds noinfarcts were observed.

Diagnosis as to the particular type of myocardial ischemic conditionrelevant to the patient can be made based on clinical signs andsymptoms, generally clinical signs or symptoms that distinguish amongconditions associated with myocardial ischemic conditions.

The assessment of risk of SAT or MI in the patient can then beincorporated into a regimen of care for the patient. For example, if apatient is above a risk threshold for SAT, then the clinician can assessthe risk and benefits of alternate therapies and further consider ananti-thrombotic regimen. If a patient is below a risk threshold for SAT,then administration of anti-thrombotics (e.g., clopidogrel) may beunnecessary or may be administered in lower doses. If a patient isborderline or abnormal, then the clinician can assess the risk andbenefits of alternate therapies and further consider an anti-thromboticregimen.

It is noted that platelet reactivity, TTF, or TFF prior to treatment(e.g., by administration of an anti-thrombotic such as clopidogrel)determines post-treatment reactivity, TTF, or TFF following coronaryartery stenting. Thus, for example, a pre-treatment platelet aggregationscore may provide an indicator of the type and extent of follow-uptherapy that is warranted.

Similarly, if a patient is above a risk threshold for myocardialischemia within 6 months, then the clinician can assess the risk andbenefits of initiating therapy or modifying a current therapy. If apatient is below a risk threshold for myocardial ischemia within 6months, then initiation or modification of therapy is not warranted. Ifa patient is borderline or abnormal then more close monitoring of thepatient is warranted.

Coordinated Diagnosis of Risk of Myocardial Ischemia Based on Profile ofMarkers: The ThromboProfile™

In one embodiment, the invention provides development of a myocardialischemia risk profile, particularly a thrombotic risk profile and/oratherosclerosis risk profile, for a patient having or suspected ofhaving vascular disease. Such a “ThromboProfile™” is used with the goalof detecting patients at risk for atherosclerosis, thrombosis, and/orbleeding in order to reduce morbidity and mortality. A “ThromboProfile™”provides a direct, immediate and accurate risk assessment available thuscircumventing estimates provided by more traditional and indirectmeasurements of thrombosis risk noted previously.

Any number of risk factors assessed by a variety of different assays canbe included in the profile analysis. Risk factors that can be includedin the myocardial ischemia risk profile include, but are not necessarilylimited to lipid risk factors (e.g., cholesterol level, LDL cholesterollevel, LDL cholesterol particle size, HDL cholesterol level, HDLcholesterol particle size, triglyceride level, LPa, Lp-PLA2, and thelike), markers of inflammatory risk factors (e.g., levels of markerssuch as, e.g., C-reactive protein, Interleukin-6, and myeloperoxidase),markers of oxidation risk factors (e.g., myeloperoxidase, oxidized LDL,oxidized fatty acids, and the like), metabolic risk factors (e.g.,glucose measurements (e.g., as measured by fasting glucose levels,hemoglobin A1C (glycosylated hemoglobin), homocysteine levels, and thelike), platelet reactivity analysis (ADP-induced aggregation (e.g., at 5μM and/or 20 μM), thromboelastography (MA), VASP-P, platelet receptors(e.g., GP IIb/IIIa, and the like), and other measures of plateletreactivity), coagulation risk factors (e.g., as assessed bythromboelastography analysis prothrombin time (PT), INR, fibrinogenlevels, platelet count, and the like) and responsiveness toanti-platelet therapy (e.g., clopidogrel response, aspirin response, andthe like), and homocysteine levels. Values provided in the table beloware merely exemplary of values for assays for these factors, whichassays and values are in the art, and which values may vary, e.g,according to differences in assays (e.g., sensitivity of assays

Desired Disease Platelet Level Level Reactivity At risk Lipid RiskFactors Total Cholesterol <200 mg/dl >200 mg/dl Aggregation >26% SAT(LTA) (5 uM) LDL Cholesterol <130 mg/dl >130 mg/dl Aggregation >45-50%(LTA) SAT (20 uM) >50% I HDL Cholesterol >40 mg · dl <40 mg/dlThrombo- >68 I elastography MA Triglycerides <150 mg/dl >150 mg/dl P2Y12React >46% LDL Particle Small dense Small dense Receptors >42 MFI SizeLDL absent LDL present LDL Particle <1100 nanomol/l >1100 nanomol/lReact time <5.1 I Concentration HDL Particle large small Coragulation<5.1 I Size Thrombo- elastography analysis (reaction time) HDLParticle >30 mg/dl <30 mg/dl Concentration LPa <10 mg/dl >10 mg/dlInflammation Risk Factors HS-CRP <1 mg/dl >1 mg/L IL-6 <1.08 pg/ml ≥2.92pg/ml ICAM-1 <267.8 ng/ml >285.2 ng/ml Metabolic Risk Factors Tailoredtherapy* Fasting glucose 70-110 mg/dl >110 mg/dl Clopidegrel HemoglobinA1C 4.5%-5.70% >6.0% Aspirin I = ischemia, SAT = subacute stentthrombosis *Tailored Therapy: Patients on clopidogrel should target 5microM < 26% or 20 microM < 45%. If not at target, consider increaseddose or alternative therapy. Aspirin aggregation by arachidonic acidtarget is: <5% for aspirin responders. If aspirin non-responsiveconsider increased dose or alternative therapy.

In one embodiment, values associated with lipid risk factor assays,inflammation risk factors, oxidation markers, and metabolic risk factorsare assessed to provide an atherosclerotic profile. In anotherembodiment, platelet reactivity assays, coagulation assays, andresponsiveness to anti-platelet drugs, are assessed to provide athrombotic profile.

In general, cholesterol levels (including total cholesterol, LDL, HDL,particle sizing), triglyceride levels, glucose measurements andhomocysteine levels assess the risk for atherosclerosis and arterialplaque formation. These indirect markers are used to assess risk ofmyocardial infraction.

The profiles generated according to the invention can be used in avariety of settings. For example, coagulation status can be used toassess the bleeding risk in patients on anticoagulants and in theperi-operative window. For instance, inhibitors of coagulation (warfarin(coumadin) and heparin) are often used as “blood thinners” and aremonitored by PT and INR.

The risk profile does not assume that patients with atherosclerosis orthrombosis are similar or should be treated in a similar manner.Instead, the patients are individually graded according to risk based onone or more of these markers. In particular, platelet reactivity,particularly platelet aggregation, is an important, yet currentlyignored, factor.

The results of assessment of various risk factors can be provided as a“scorecard” as exemplified above. In one embodiment, one or more assaysare conducted and provided on such a scorecard. In another embodiment,the scorecard is provided as a display on a device (e.g., handhelddevice, computer desktop, website) that results from execution of acomputer program to provide an electronic or “virtual” scorecard, asdisplayed above.

Manual and Computer-Assisted Methods and Products Computer Program

The values from the assays described above, such as the the plateletreactivity score (e.g., platelet aggregation score orthromboelastography MA score), TTF score, and TFF score (e.g., where TTFand TFF are assessed by thromboelastography R), can be calculated andstored manually, e.g., on a physical scorecard as exemplified above.Alternatively, the above-described steps can be completely or partiallyperformed by a computer program product. The present invention thusprovides a computer program product including a computer readablestorage medium having a computer program stored on it. The program can,when read by a computer, executes relevant calculations based on valuesobtained from analysis of one or more biological sample from anindividual (e.g., of changes in values associated with therapy (e.g., apretreatment value vs. a posttreatment value and/or values obtained attimes t₁ and t₂ over a selected period, e.g., during administration of atherapeutic regimen), conversion of values from assays to a score aboveor below a selected threshold, and the like) The computer programproduct has stored therein a computer program for performing thecalculation.

The computer program product can also analyze the data, as depicted inFIG. 20, wherein a value for an assay result is determined; and thecomputer program product then determines whether the value is above orbelow a pre-determined value, e.g., a risk threshold value. For example,in the context of assessing risk of a thrombotic event by assessingplatelet aggregation, a platelet aggregation score or value is comparedto a risk threshold value, e.g., 50% 5 μM ADP-induced plateletaggregation (e.g., as assessed by LTA). Based comparison to thethreshold value, the computer program product assesses the risk ofthrombosis in the patient, and displays a result to the user. The valuesfor analysis by the computer program can be input into the programmanually, or the values can be obtained from an assay device (e.g., thevalues obtained by a light transmission aggregometer (LTA) device) canbe transferred directly into the program so as to avoid manual input.

In other embodiments, the computer program is designed to store valuesfor 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different assays associatedwith the ThromboProfile™ described above. In one embodiment, and in amanner similar to that described above in the context of a plateletaggregation assay, the program analyzes one or more of these assayvalues to determine whether the value is above or below a pre-determinedvalue, e.g., a threshold value, such as a risk threshold value. Basedcomparison of an assay value to the corresponding threshold value forthat assay, the computer program product assesses the risk in thepatient, and displays a result to the user. The values for analysis bythe computer program can be input into the program manually, or thevalues can be obtained from an assay device.

Exemplary assay values which can be included in the invention includevalues obtained from assay(s) of one or more of lipid risk factors(e.g., total cholesterol, LDL cholesterol, HDL cholesteroltrigylcerides, LDL particle size, HDL particle size, LDL concentration,HDL particle size, HDL particle size, LPa, Lp-P)LA2, and the like),inflammation risk factors (e.g., HS-CRP, IL-6, ICAM-6), oxidationmarkers (e.g., myeloperoxidase, oxidized LDL, oxidized fatty acids, andthe like), metabolic risk factors (fasting glucose, hemoglobin A1C, andthe like), platelet reactivity (e.g., aggregation (e.g., by 5 μM ADP-and/or 20 μM ADP-induced aggregation), thromboelastography analysis,VASP-P, platelet receptors (e.g., GP IIa/IIIb and the like), and othermeasures of platelet reactivity), coagulation (e.g., thromboelastographyanalysis and the like), and responsiveness to anti-platelet therapy(e.g., clopidogrel response, aspirin response, and the like). In oneembodiment, values associated with lipid risk factor assays,inflammation risk factors, oxidation markers, and metabolic risk factorscan be displayed separately as values relating to risk ofatherosclerosis. Similarly, values associated with platelet reactivityassays, coagulation assays, and responsiveness to anti-platelet drugs(e.g., clopidogrel, aspirin, and the like) can be displayed separatelyas risk factors for thrombosis.

Optionally, the program can store multiple values from multipledifferent assays and/or from the same assays conducted at differenttimes. The program can also store multiple risk assessment calculationresults so as to provide a picture of the patient's thrombotic risk overtime (e.g., during a course of therapy). In addition, the program canstore any of a variety of patient information details (e.g., patientvitals and statistics, treatment history (particularly with respect totherapy administered at the time of an assay for which an assay valueand/or risk assessment calculation is stored), and the like).

Systems and Apparatus

In a related embodiment, the invention provides a system for executingthe program described above, which system generally includes: a) acentral computing environment; b) an input device, operatively connectedto the computing environment, to receive patient data, wherein thepatient data can include, for example, a platelet aggregation score orvalue or other value obtained from an assay using a biological samplefrom the patient as described in detail above; c) an output device,connected to the computing environment, to provide information to a user(e.g., medical personnel); and d) an algorithm executed by the centralcomputing environment (e.g., a processor), where the algorithm isexecuted based on the data received by the input device, and wherein thealgorithm calculates a risk of a thrombotic event, e.g., by comparing aplatelet aggregation score to a threshold value associated with risk ofthrombosis, including risk of acute myocardial infarction, where aplatelet aggregation score above the threshold value indicates risk ofthrombosis within a period in the future, e.g., within 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 months, usually within 4 monthsto 12 months, more usually within 6 months to 12 months, still moreusually within 6 months from the time of the assay.

The instant invention further provides a portable apparatus having acomputer readable medium (e.g., a processor) that stores data,calculates risk based on the algorithm described above, and provides anassessment of risk of a thrombotic event based on the calculation. Theportable apparatus can also store multiple values and risk assessmentcalculation results so as to provide a picture of the patient'sthrombotic risk over time (e.g., during a course of therapy).

In some embodiments, a subject apparatus (e.g., a portable apparatus)comprises: a) a device for receiving and storing patient data asdescribed above, including assay values, calculation results, andpatient information; b) a data output device; and c) an algorithm storedwithin the computer program product within the apparatus, whichalgorithm, for example, assesses risk of a thrombotic event based on aplatelet aggregation score compared to a risk threshold value, whichinformation is transmitted to the data output device, where the outputdevice displays the information (e.g., “high risk of thrombosis” or “lowrisk of thrombosis”) to a user.

The data input device (also referred to as an operator input device) maybe, e.g., a keyboard, a mouse, and the like. The processor has access toa memory, which may be any suitable device in which the processor canstore and retrieve data, such as magnetic, optical, or solid statestorage devices (including magnetic or optical disks or tape or RAM, orany other suitable device). The processor can include a general purposedigital microprocessor (such as is typically used in a programmablecomputer) suitably programmed to execute an algorithm as describedabove, or any hardware or software combination which will perform therequired functions.

In some embodiments, the processor will be programmed to calculate therisk of thrombosis based on a platelet aggregation score obtained from abiological sample from an individual. The information will betransmitted to the output device for display to a user. The informationwill in some embodiments be displayed as “high risk of thrombotic event”or “low risk of thrombotic event,” although alternative language ispossible.

In some embodiments, the portable apparatus comprises: a) a device fordetermining an assay value (e.g., a platelet aggregation score) from abiological sample; b) a device for communicating (e.g., transmitting)the determined value to the receiving and storage device; c) a devicefor receiving and storing patient data, where the data can include, forexample, the age of the patient, the gender of the patient, priorcardiac history, history of therapy (including percutaneous andnon-percutaneous (e.g., drug) intervention) and values obtained fromassays conducted with a biological sample from the patient; d) a dataoutput device; and e) an algorithm stored within a computer programproduct within the apparatus, which algorithm is executed to, forexample, determine whether a platelet aggregation score or value isabove or below a risk threshold value; and assess risk of a thromboticevent in the patient. The result is transmitted to the data outputdevice, where the output device displays the assessment to a user, whichassessment can optionally include the platelet aggregation value, therisk threshold value used, or both. Suitable devices for determining avalue for an assay results for the assays described above are well knownin the art. For example, where the assay is an ADP-induced plateletaggregation assay, the device can be a light transmission aggregometer.

In general, a subject apparatus will include a computer readable mediumincluding the programming described above. The computer program can berecorded on computer readable media, e.g., any medium that can be readand accessed directly or indirectly by a computer. Such media include,but are not limited to: magnetic tape; optical storage such as compactdisc-read only memory (CD-ROM) and digital versatile disk (DVD);electrical storage media such as random access memory (RAM) andread-only memory (ROM); and hybrids of these categories such asmagnetic/optical storage media. One of skill in the art can readilyappreciate how any suitable computer readable media can be used tocreate a manufacture that includes a recording of the presentprogramming/algorithms for carrying out the above-described methodology.In certain embodiments, the programming is further characterized in thatit provides a user interface, where the user interface presents to auser the option of selecting among one or more different, includingmultiple different, criteria, e.g., age of individual, etc. Theinstructions may include installation or setup directions. Theinstructions may include directions for use of the invention.

In addition, a subject apparatus will typically include instructions forusing the apparatus to carry out a subject method. The instructions ofthe above-described apparatus are generally recorded on a suitablerecording medium. For example, the instructions may be printed on asubstrate, such as paper or plastic, etc. As such, the instructions maybe present in the apparatus as a package insert, or components thereof(i.e. associated with the packaging or sub packaging), etc. In otherembodiments, the instructions are present as an electronic storage datafile present on a suitable computer readable storage medium, e.g.,CD-ROM, diskette, etc, including the same medium on which the program ispresented.

In yet other embodiments, the instructions are not themselves present inthe apparatus, but means for obtaining the instructions from a remotesource, e.g. via the Internet, are provided. An example of thisembodiment is an apparatus that includes a web address where theinstructions can be viewed and/or from which the instructions can bedownloaded. Conversely, means may be provided for obtaining the subjectprogramming from a remote source, such as by providing a web address.Still further, the apparatus may be one in which both the instructionsand software are obtained or downloaded from a remote source, as in theInternet or World Wide Web. Some form of access security oridentification protocol may be used to limit access to those entitled touse the subject invention. As with the instructions, the means forobtaining the instructions and/or programming is generally recorded on asuitable recording medium.

Tailoring of Therapy

In one embodiment, the invention features use of the methods andcompositions described herein in the context of tailoring therapy.Analysis of one or more risk factors for myocardial ischemia asdescribed above can be used to provide information to the clinician asto the responsiveness of a patient to therapy. Assessment of plateletreactivity (particularly platelet aggregation) in tailoringanti-platelet therapy is of particular interest, and can be conducted inconcert with assessment of one or more additional risk factors of athrombotic risk profile and/or atherosclerotic risk profile as describedabove.

The invention can be used to aid selection of therapy and to assess andfollow a selected therapy. Therapies for myocardial ischemic conditionsinclude drug-based therapies (e.g., pharmacological or adjuvanttherapies), mechanical therapies (e.g., surgical or non-surgicalintervention associated with physical manipulation of tissue, e.g.,stenting, angioplasty, grafting, and the like), or a combination ordrug-based and mechanical therapy.

Of particular interest is assessment of efficacy (e.g., in reducing riskof thrombosis) of therapy involving administration of one or moreanticoagulant or antithrombotic drugs. Anticoagulant drugs includereversible or irreversible anti-platelet inhibitors such as aspirin,glycoprotein IIb/IIIa (GP IIb/IIIa) inhibitors (e.g., abciximab (RePro™)(a chimeric monoclonal antibody), tirofiban (Aggrastat™, an RGDpeptidomimetic), and eptifibatide (Integrelin™, a small peptide)),ADP-dependent aggregation inhibitors (e.g., clopidogrel, ticlopidine),and the like

In particular, eptifibatide has demonstrated efficacy in the treatmentof patients during coronary angioplasty, myocardial infarction andangina, and is indicated for administration in acute coronary syndromes(ACS) including acute myocardial infarction (AMI).

Anti-thrombotic drugs for which efficacy can be assessed, and therapymonitored, according to the invention include low molecular weightheparins (e.g., enoxaparin (Lovenox)), and direct antithrombotics suchas hirudin, hirulog (bivaluridin), and argatroban.

Efficacy of mechanical intervention (including percutaneous ornon-invasive interventions) can also be assessed using the methods andcompositions of the invention. Examples of mechanical interventionsinclude angioplasty, stenting (e.g., coronary stent), atherectomy, laserangioplasty, brachytherapy, percutaneous myocardial revascularization(PMR), intravascular ultrasound, and balloon valvuloplasty.

Efficacy of therapy can be assessed by examining improvement in one ormore clinical symptoms of disease. Successful therapy is normallyconsidered to be a significant improvement in one or more clinicalsymptoms after treatment.

An aspect of the invention of particular interest relates to assessing apatient's platelet reactivity (particularly by assessing plateletaggregation), assessing a patient's responsiveness to anti-platelettherapy, and tailoring therapy to suit a patient's platelet reactivityand/or responsiveness to anti-platelet therapy. As discussed above, theinventor has found that high platelet reactivity is associated with ahigh risk of an ischemic event, e.g., a thrombotic event, regardless ofwhether that platelet reactivity is responsive to therapy (e.g.,regardless of whether platelet reactivity is reduced in response toadministration of drug, e.g., an anti-platelet inhibitor such asclopidogrel).

Individual patients have variable response to anti-platelet medications.Furthermore, patients with low responsiveness to therapy (e.g., patientsexhibiting drug resistance) may be at risk for complications (e.g.,thrombosis). Moreover, this inter-individual variability inresponsiveness to platelet inhibition has been clearly demonstrated inpatients with atherosclerosis of the coronary arteries who haveundergone coronary artery stenting. In these patients, it is desirableto give them anti-platelet medication to prevent thrombosis. Theanti-platelet drug, clopidogrel (Plavix™) is most commonly used toprevent thrombosis in a standard amount.

As discussed herein, non-responder patients usually have high plateletreactivity and hence, greater risk of thrombosis. Conversely, patientswith low platelet reactivity have a small chance of thrombosis and seemto respond to even small amount of clopidogrel. Patients range fromresponsive, partially responsive, and non-responsive to clopidogreltherapy. In addition, another anti-platelet drug, aspirin, is associatedwith non-responsiveness.

According to the invention, patients with high platelet reactivity meritclose monitoring and may require adjustment of anti-platelet regimen(e.g., adjustment of dose, dosing schedule, or drug). Alternatively,patients with low platelet reactivity need little or no medications.Tailoring therapy according to the invention thus avoids administrationof drug to those patients who are at lower risk of complications (e.g.,thrombosis) and/or allows the clinician to select a dosing regimen thatavoids administration to an amount of drug that is greater than what isneeded. In short, dose titration of drug can lead to a reduction inmorbidity and mortality. Thus the invention helps reduce the incidenceof side effects associated with drug-based therapy, as well as providingcost-savings.

Since most US adults take aspirin for cardiovascular prophylaxis, theimplications are even more far-reaching. Indeed, stroke patients andpatients with a tendency to suffer from blood clots (e.g.,hypercoagulable state, coach class syndrome, women on birth controlpills, patients with pulmonary embolism, etc.) would also benefit fromsuch knowledge. Finally, in the peri-operative state where most patientsare placed on anti-platelet therapy (intra-abdominal surgeries,orthopedic procedures, cardio thoracic operations, etc.); knowledge ofplatelet reactivity and hypercoagulability becomes very important indetermining the need for and selection of therapy. Indeed, as morepotent anti-platelet agents are developed, the ability to assess risk ofthrombosis is of increased importance.

Newer and more powerful antiplatelet agents that target the sameplatelet receptor as clopidogrel are being developed. The ability tomeasure how an individual patient will respond to these medications in areliable fashion is important. Undoubtedly, adjustment of thesemedications will reduce complications, enhance patient care and lead tocost-savings.

Treatment Regimen Modification

Modification of a treatment regimen includes one or more of: modifying(increasing or decreasing) the dosing frequency of the active agentadministered; administering one or more additional active agents;modifying (increasing or decreasing) the amount of active agentadministered; and administering a different active agent from the activeagent, e.g., discontinuing administration of the active agent.

In some embodiments, modifying a dosing regimen comprises increasing thedosing frequency, e.g., increasing administering the active agent fromonce per week to twice per week, to three times per week, to daily, orto twice daily. Thus, e.g., in some embodiments, a method comprisingadministering an active agent at a frequency of once per week ismodified such that the active agent is administered twice per week,three times per week, daily, or twice daily.

In some embodiments, modifying a dosing regimen comprises increasing theamount of active agent administered, e.g., increasing the amount ofactive agent administered over a given time period by at least about25%, at least about 50%, at least about 100% or 2-fold, at least about2.5-fold, at least about 3-fold, at least about 4-fold, or at leastabout 5-fold, or more. Thus, e.g., in some embodiments, a methodcomprising administering an active agent at a first dose over a giventime period is modified such that the active agent is administered at asecond dose that is at least about 25%, at least about 50%, at leastabout 100% or 2-fold, at least about 2.5-fold, at least about 3-fold, atleast about 4-fold, or at least about 5-fold, or more, higher, over thesame time period. As one non-limiting embodiment, where the active agentis administered at a dose of 100 μg TIW, and assessment of thromboticrisk indicates that this treatment regimen is not efficacious, thetreatment regimen is modified to comprise administering 200 μg of theactive agent TIW.

In some embodiments, a further risk assessment step is performed afterthe therapy modification step, to assess the efficacy of the modifiedtreatment regimen. A further modification of the modified treatmentregimen will in some embodiments be carried out.

In some embodiments, modifying a dosing regimen comprises administeringat least a second active agent, and, in some embodiments comprisesdiscontinuing administration of the first active agent. For example, inthe context of anti-platelet therapy it may be desirable to administertwo different types of anti-platelet drugs (e.g., an ADP-inducedaggregation inhibitor (e.g., clopidogrel) and a GP IIb/IIIa inhibitor(e.g., eptifibatide)).

Kits

Kits including one or more of compositions for conducting an assay for arisk factor described herein, with reagents for a platelet aggregationassay. In addition to the compositions, the kits include aninformational or instructional package insert describing the assay to beconducted and, in the case of a platelet aggregation assay, informationrelating to a risk threshold value for platelet aggregation, asdescribed above. The instructions can be printed on a label affixed tothe container, or can be a package insert that accompanies thecontainer.

For example, the kit includes a chart to facilitate assessment ofplatelet aggregation and the associated risk of a thrombotic event. Inanother embodiment, the kit includes a scorecard for recording resultsof various assays to provide a “profile” or ThromboProfile™ of thepatient's risk of thrombosis and/or atherosclerosis. In anotherembodiment, the kit includes a handheld device which is preprogrammed toreceive one or more assay result values and/or to determine whether aplatelet aggregation value is above or below a risk threshold value. Thedevice can optionally provide a readout indicating an overall riskassessment and/or a risk assessment based on a platelet aggregationscore.

Instructions for practicing the subject methods are generally recordedon a suitable recording medium. For example, the instructions may beprinted on a substrate, such as paper or plastic, etc. As such, theinstructions may be present in the kits as a package insert, in thelabeling of the container of the kit or components thereof (i.e.,associated with the packaging or subpackaging) etc. In otherembodiments, the instructions are present as an electronic storage datafile present on a suitable computer readable storage medium, e.g.CD-ROM, diskette, etc. In yet other embodiments, the actual instructionsare not present in the kit, but means for obtaining the instructionsfrom a remote source, e.g. via the internet, are provided. An example ofthis embodiment is a kit that includes a web address where theinstructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric.

Example 1: Study of Platelet Reactivity in Patients with and withoutStent Thrombosis: Clopidogrel Resistance and Stent Thrombosis (CREST)Methods and Materials

Patients and Blood Samples.

Cases of stent thrombosis (n=30) were identified by searching themedical records of patients who underwent coronary stenting in the last1.5 years at the Sinai Hospital of Baltimore and Union MemorialHospital, Baltimore, Md. This study was approved by the InvestigationalReview Board at the hospitals. Stent thrombosis was defined by thesudden onset of coronary artery occlusion in a stented vessel resultingin hospitalization and judged by the treating interventionalist as dueto thrombosis. Platelet reactivity in patients with stent thrombosis(SAT) was compared to a group of patients without SAT. In patientswithout SAT, platelet studies were performed 5-14 days post-procedureand these patients were enrolled consecutively (n=100).

In patients with SAT, the average time from the occurrence of stentthrombosis to the initial blood drawn for laboratory evaluation was218±204 days. In patients with SAT already receiving a maintenance doseof clopidogrel (75 mg·qd), blood was studied on the day of arrival atthe Center. Those patients (n=2) not receiving clopidogrel at the timeof the study were reloaded with 300 mg; maintained on 75 mg daily; andreturned for blood draw 5 days later. Blood was drawn with a 21 g needleand placed into vacutainer blood collecting tubes (Becton-Dickinson,Rutherford, N.J.) containing 3.8% trisodium citrate after discarding thefirst 2-3 ml of free flowing blood. The vacutainer tube was filled tocapacity and gently inverted 3 to 5 times to ensure complete mixing ofthe anticoagulant. Patients identified who were in-hospital had blooddrawn at day 5 post-procedure. No patients in the study had beenreceiving Glycoprotein IIb/IIIa (GP IIb/IIIa) inhibitors oranticoagulants within 96 hours of blood sampling. All patients werereceiving aspirin (81-325 mg qd) except two patients in SAT group.

Platelet Reactivity Measurements Platelet Aggregation.

Platelet aggregation was determined by conventional light transmittanceaggregometry in response to 5 and 20 μM ADP (adenosine diphosphate); and1.0 mM arachidonic acid using standard methods as previously described.Briefly, the blood citrate mixture was centrifuged at 120×g for 5 min torecover the platelet-rich plasma (PRP) and further subjected tocentrifugation at 850×g for 10 min to recover platelet-poor plasma(PPP). The PRP and PPP were stored at room temperature for use within 2h. The platelet count was determined in the PRPR sample and adjusted to2-3×10⁸/ml with homologous PPP. Platelets in PRP were stimulated with afinal concentration of 5 and 20 μM ADP (Chronolog, Haverton, Pa.), andpercent aggregation was assessed as previously described using aChronolog Lumi aggregometer (Model 490A) with the Aggro-Link softwarepackage (Gurbel et al. J Am Coll Cardiol 1998 89:783).

GP IIb/IIIa Receptors.

The surface expression of platelet receptors was determined by wholeblood flow cytometry using a multicolor analysis method (ImmunocytometrySystems, Cytometry Source Book, Becton Dickinson) with the followingmonoclonal antibodies: FITC (fluorescein isothiocyanate—conjugated)PAC-1 (recognizes the active GP IIb/IIIa receptor) and R-phycoerythrin(R-PE) conjugated CD41a (recognizes the total GP IIb/IIIa receptorpopulation). Antibodies were obtained from BD Biosciences, San Diego,Calif. The blood-citrate mixture was stimulated with 5 μM ADP for 2minutes. Saturating concentrations of respective antibodies were thenadded to unstimulated and simulated blood and the tubes were incubatedat room temperature for 20 minutes in the dark. The labeled samples werefixed by the addition of 1% buffered paraformaldehyde and stored at 4°C. for at least 2 hours. The labeled samples were analyzed by a BectonDickinson FACScan flow cytometer set up to measure fluorescence lightscatter that was calibrated daily with fluorescence beads for themulticolor flow cytometer setup (CaliBRITE™3, BD Biosciences). Aftersetting the gate around platelets, FL1(FITC)/FL2(R-PE) compensationswere adjusted. All the variables were collected by use of 4-decadelogarithmic amplification. The data were collected in list mode and thenanalyzed using CELL Quest Software (BD Biosciences). Total and activatedGP IIb/IIIa receptor levels were expressed as log MFI (Gurbel et al.Thromb Res 2003; 112:9-12).

Vasodilator Stimulated Phosphoprotein (VASP).

The phosphorylation of VASP is a marker of P2Y₁₂ receptor reactivity andthus, clopidogrel-induced inhibition (Barragan et al. CatheterCardiovasc Interv. 2003; 59:295-302). Stimulation of platelets by ADPleads to Gi-coupled inhibition of adenylate cyclase which, in turnreduces protein kinase activity and VASP phosphorylation levels. VASPphosphorylation levels were quantified with labeled monoclonalantibodies by flow cytometry using the Platelet VASP-FCM kit (BiocytexInc, Marseille, France). The P2Y₁₂ reactivity ratio is calculated aftermeasuring the VASP phosphorylation levels following stimulation withPGE₁ (MFI PGE₁) and PGE₁+ADP (MFI PGE₁+ADP). The P2Y₁₂ reactivityratio=[(MFI PGE₁)−(MFI PGE₁+ADP)/(MFI PGE₁)]×100%. Thus, the lower theratio, the higher the clopidogrel induced inhibition of the P2Y₁₂receptor.

Clopidogrel Resistance.

Clopidogrel resistance for purposes of this study was defined as>75_(th) percentile for 5 and 20 μM ADP-induced aggregation as measuredin the group without SAT. Aspirin resistance was defined as >14% changein light transmittance after stimulation with arachidonic acid (Gum etal. J Am Coll Cardiol. 2003; 4:961-5).

Statistical Analysis.

Comparisons were made between groups by one way analysis of variance(Statistica software, Tulsa, Okla.). Standard regression analysis wasused to correlate aggregation with the other markers measured.(Statistica software, Tulsa, Okla.). The Wilks-Shapiro test was used toassess conformity with a normal distribution. Based on the normaldistribution of data, the mean±SD and mean±SE were used and P<0.05 wasconsidered significant.

Results

Patients.

A total of 5355 interventional coronary procedures were performed at the2 hospitals over an 18 month period and thirty patients (0.6%) wereidentified as having SAT. Among these patients, there were 3 deaths; 5patients could not be contacted; 2 did not participate because ofclopidogrel allergy (skin rash); and the remaining twenty patientsagreed to participate in the study. Among these 20 patients, 18 werereceiving a 75 mg maintenance dose of clopidogrel and 2 patients wereretreated with clopidogrel. 18/20 SAT patients were on aspirin therapy(81-325 mg per day). The mean time to SAT (time from the day ofprocedure to the development of SAT) was 23±16 days.

Demographics.

The clinical and angiographic characteristics of both groups are shownin Tables 1 and 2, respectively. All of the non-SAT interventions wereperformed electively whereas 12 (60%) of procedures resulting in SATwere performed emergently. The ages of both groups were the same.Patients with SAT had a non-significantly greater incidence of familyhistory of CAD. Hematological data did not differ between groups. Theejection fraction was lower and the total lesion length was greater inthe SAT group.

TABLE 1 Patient Demographics SAT No SAT (n = 20) (n = 50) p - value Age(years) 65 ± 11 62 ± 13 NS Race (Caucasian) n, (%) 10 (50) 38 (76) .04Gender (Male) n, (%) 10 (50) 35 (70) NS Risk Factors/Past medical Hx n,(%) Smoking 9 (45) 27 (54) NS Family history of CAD 16 (80) 28 (56) .06Hypertension 15 (75) 28 (56) NS Hyperlipidemia 15 (75) 36 (77) NSDiabetes 12 (60) 21 (42) NS Prior Myocardial Infarction 12 (60) 24 (48)NS Prior CABG 5 (25) 12 (24) NS Prior PTCA 4 (20) 20 (40) NS BaselineMedications n, (%) Beta blockers 17 (85) 45 (90) NS ACE Inhibitors 15(75) 26 (52) NS Calcium blockers 5 (25) 10 (20) NS Lipid lowering agents3A4 11 (55) 29 (58) NS Non 3A4 5 (25) 13 (26) NS CABG = coronary arterybypass graft surgery; CAD = coronary artery disease; PTCA = percutaneouscoronary angioplasty; ACE = angiotensin converting enzyme; SAT = stentthrombosis

TABLE 2 Procedural Characteristics SAT No SAT (n = 20) (n = 50) p ValueEjection Fraction (%) 40 ± 10 53 ± 6   .001 Number of vessels treated1.3 ± 6.4 1.4 ± 0.7 NS Lesion Morphology Denovo n, (%) 20 (100) 46 (92)NS Lesion Location n, (%) LAD 5 (30) 14 (28) NS CX 3 (15) 11 (22) NS RCA12 (60)  17 (34) .05 SVG 0 (0)   8 (16) NS Stent Types Drug eluting n,(%) 6 (30) 31 (62) .01 Bare metal n, (%) 14 (70)  16 (32) .01 Referencevessel diameter (mm) 2.8 ± 0.3 3.1 ± 0.4 NS Total lesion length (mm) 24± 6  18 ± 8   .007 Pre-stenosis (%) 94 ± 7  87 ± 12 NS Post-stenosis (%) 9 ± 12 3 ± 6 NS CX = circumflex artery; LAD = left anterior descendingartery; RCA = right coronary artery; SVG = saphenous vein graft

Platelet Data.

Platelet aggregation in response to 5 and 20 μM ADP was higher in thegroup with SAT as compared to the group without SAT (FIGS. 1 and 2,p<0.05 for both 5 μM and 20 μM ADP-induced aggregation). Patients withSAT had greater active GP IIb/IIIa expression and a higher P2Y₁₂reactivity ratio whereas total GP IIb/IIIa expression was notsignificantly different between groups (Table 3). CD41, which is ameasure of total receptor expression, served as a control.

TABLE 3 Platelet Characteristics No SAT SAT p Value LTA - 5 μM ADP (%)26 ± 2 49 ± 4 <0.001 LTA - 20 μM ADP (%) 46 ± 2 65 ± 3 <0.001 LTA-Arachidonic Acid 0 non-responder 1 non-responder NS P2Y₁₂ReactivityRatio 46 ± 9 69 ± 5  0.030 GP IIb/IIIa (MFI) Unstimulated 15 ± 3  9 ± 1NS Stimulated 42 ± 4 138 ± 19 <0.001 CD 41 (MFI) Unstimulated 513 ± 30515 ± 31 NS Stimulated 729 ± 60 770 ± 38 NS LTA = light transmittanceaggregometry; MFI = mean fluorescence intensity; NS = not significant

The estimated incidence of clopidogrel resistance was 65% and 60% asmeasured by 5 μM and 20 μM ADP-induced aggregation, respectively (FIGS.1 and 2). The r value comparing 5 μM ADP-induced aggregation to 20 μMADP-induced aggregation was 0.93. The reactivity ratio by VASP assaycorrelated strongly with 20 μM ADP-induced aggregation (r=0.57). Thecorrelation of stimulated active GP IIb/IIIa expression with aggregationwas weaker (r=0.36 with 5 μM ADP-induced aggregation and 0.29 with 20 μMADP-induced aggregation, respectively). All patients were responsive toaspirin except 1 in the SAT group (54% aggregation). Arachidonic acidinduced aggregation (1 mM) was 4±2% in the non-SAT group vs. 3±2% in theSAT group (p=NS).

The findings above demonstrate a strong association between heightenedplatelet reactivity, particularly as detected by platelet aggregation,low responsiveness to clopidogrel, and the development of stentthrombosis.

Based on these data, platelet aggregation below about 40% for 20 μM ADPis associated with a 0% incidence of SAT. Importantly, the data fromthis study indicate that platelet reactivity alone, particularly asmeasured by ADP-induced platelet aggregation, is a predictor of risk ofSAT. Platelet aggregation in patients without SAT was assessed at 5-14days post-procedure (e.g., post-stent). Of those who experienced SAT theaverage time from this initial blood draw to occurrence of stentthrombosis was 218±204 days, or from about 6 months to about 14 months.

The 0.6% incidence of SAT in this study is in agreement with previouslyreported data indicating the incidence of subacute stent thrombosis tobe in the range of 0.4% to 3% in high risk patients, with an incidenceof 0.4% in 500 patients treated with a sirolimus-eluting stent (Reynoldset al. J Invasive Cardiol. 2002; 14:364-8; Regar et al. Am J Cardiol.2004; 93:1271-5). Most of the patients with SAT in this study had theirindex procedure performed emergently and had long lesions. These riskfactors are also supported by other studies (Reynolds et al. J InvasiveCardiol. 2002; 14:364-8; Regar et al. Am J Cardiol. 2004; 93:1271-5).

The results above illustrates that the variability in final plateletreactivity in a patient given the standard dose of clopidogrel. In thepresent study, platelet aggregation was measured using two agonistconcentrations. There was a strong correlation between aggregationinduced by both agonist concentrations. In addition, platelet receptorexpression was significantly greater in the SAT group. These resultsdemonstrate that irrespective of the methodology chosen to measureplatelet reactivity (e.g., light transmittance aggregometry, active GPIIb/IIIa, or VASP-phosphorylation levels, or thromboelastography), theresponse to clopidogrel therapy is indeed heterogeneous, and patientswith stent thrombosis have greater platelet reactivity to ADP thanpatients without stent thrombosis.

The VASP assay is a direct measure of P2Y₁₂ reactivity and thereforedirectly assesses the intrinsic functional response of the receptor inthe presence of clopidogrel. The results above strongly suggest that theP2Y₁₂ receptor is not adequately inhibited in a large percentage ofpatients who have experienced SAT.

Clopidogrel resistance has been previously defined as less than 10%absolute change in aggregation compared to a pretreatment baseline(Gurbel et al. Circulation. 2003; 107:2908-13). In contrast, the studypresented herein a baseline measurement of platelet reactivity was notdetermined, as this study was not prospective with respect to the SATgroup. Given the overall low event rate of SAT, the number of patientsrequired to assess the relation of platelet reactivity to SAT in aprospective investigation was prohibitively large. Therefore,clopidogrel resistance was defined as a response higher than the>75_(th) percentile measurement with the respective marker in thepatients without SAT.

Although there is no uniformity in the definition of aspirin resistance,various measurements of platelet function in patients receiving aspirinhave been correlated to a greater risk of cardiovascular events. Theprevalence of aspirin resistance has been reported between 5-45% (Masonet al. Rev Cardiovasc Med. 2004; 5:156-163; Eikelboom et al.Circulation. 2002; 105:1650-5; Zimmermann et al. Circulation. 2003;108:542-7). Of interest, in the present study platelet reactivity wasvery low in response to 1.0 mM arachidonic acid. These results arediscordant with observations by other investigators (Gum et al. J AmColl Cardiol. 2003, 4:961-5; Eikelboom et al. Circulation. 2002;105:1650-5). Gum et al estimated aspirin resistance by using acombination of responsiveness to both ADP and a slightly higher dose ofarachidonic acid (1.6 mM). In their study mean aggregation in responseto arachidonic acid was 11.4±10.3% as compared to ˜4% in the presentstudy. The lower arachidonic acid concentration in the present study mayin part explain why only 1/70 (1.5%) met the criteria for resistance ascompared 5.5% in the Gum et al. study. The effect of aspirin to blockcyclooxygenase activity is likely much more uniform whereas the effecton platelet aggregation as measured by agonists other than arachidonicacid is influenced by many factors. Therefore, aspirin resistancesecondary to a lack of inhibition of cyclooxygenase is probably rare asreflected in the present study.

Limitations.

Platelet studies were performed at different times in non-SAT and SATpatients. Non-SAT patients were enrolled and studied prospectivelywhereas patients with SAT were identified retrospectively andsubsequently studied. The analyses of platelet reactivity conducted atdifferent intervals from the index procedure may affect the results.However, it has been previously demonstrated that clopidogrelresponsiveness is lowest early after stenting (Gurbel et al.Circulation. 2003; 107:2908-13). Therefore, this fact would onlystrengthen the discovery here, since at a later date it would beexpected that the non-SAT patients would have even higher clopidogrelresponsiveness. In conclusion, the current study indicates that highplatelet reactivity and low clopidogrel responsiveness are risk factorsfor SAT.

Example 2: Clopidogrel Loading with Eptifibatide to Arrest theReactivity of Platelets Methods

This study was approved by the Investigational Review Board. Consecutivepatients undergoing elective coronary stenting were enrolled aftergiving informed consent. Patients were >18 years old. The exclusioncriteria were: a history of bleeding diathesis, acute myocardialinfarction within 48 hours, elevated cardiac markers (above upper limitsnormal for the respective assay), cerebrovascular event within 3 months,chronic vessel occlusion or angiographically visible thrombus, illicitdrug or alcohol abuse, prothrombin time greater than 1.5 times control,platelet count <100,000/mm₃, hematocrit <30%, creatinine >4.0 mg/dl, andthienopyridine or glycoprotein (GP) IIb/IIIa use prior to the procedure.

Patients were randomly assigned to one of four treatment regimens by acomputer generated assignment that was chosen from a sealed envelope bythe study personnel: Group A) clopidogrel (300 mg); Group B) clopidogrel(600 mg); Group C) clopidogrel (300 mg+eptifibatide); and Group D)clopidogrel (600 mg)+eptifibatide. The clopidogrel loading dose wasgiven to all patients immediately after stenting and was followed by 75mg daily. In addition, all patients had received at least 81 mg aspirinfor 7 days prior to the procedure (>90% received 325 mg) and 325 mg wasadministered on the day of the procedure and daily thereafter.Eptifibatide was administered using the ESPRIT study protocol as adouble bolus (180 μg/kg) followed by an infusion (2 μg/kg/min) for 18-24hours post procedure. Unfractionated heparin was administered accordingto the ESPRIT dosing regimen (60 U/kg) as a bolus to all patients in thecatheterization laboratory immediately prior to stenting.

Blood Sampling.

Baseline blood samples were obtained in the catheterization laboratorythrough the indwelling femoral vessel sheath and transferred tovacutainer blood collecting tubes (Becton-Dickinson, Rutherford, N.J.)containing 3.8% trisodium citrate after discarding the first 2-3 ml offree flowing blood. The vacutainer tube was filled to capacity andgently inverted 3 to 5 times to ensure complete mixing of theanticoagulant. Samples were obtained before clopidogrel, eptifibatideand heparin administration (baseline); and at 3 hours, 8 hours, and18-24 hours post-stenting. The 18-24 hour blood draw was performed atthe time of completion of the eptifibatide infusion.

Platelet Aggregation.

The blood-citrate tubes were centrifuged at 120 g for 5 minutes torecover platelet rich plasma (PRP) and further centrifuged at 850 g for10 minutes to recover platelet poor plasma (PPP). The platelet count wasdetermined in the PRP sample and adjusted to 3.0×10₈/ml with homologousplatelet poor plasma. The PRP and PPP were stored at room temperature tobe used within two hours. Platelet aggregation was assessed as describedpreviously. Briefly, platelets were stimulated with 5 and 20 μM ADP andthe aggregation was assessed using a Chronolog Lumi-Aggregometer (Model490-4D) with the aggregolink software package (Chronolog, Havertown,Pa.). Aggregation was expressed as the maximum percent change in lighttransmittance from baseline, using PPP as a reference.

Whole Blood Flow Cytometry.

The surface expression of platelet receptors was determined by wholeblood flow cytometry using three-color analysis method (ImmunocytometrySystems, Cytometry Source Book, Becton Dickinson) with the followingmonoclonal antibodies: FITCconjugated PAC-1 (recognizes activated GPIIb/IIIa receptors), R-Phycoerythrin (R-PE)-conjugated CD41a (recognizestotal GP IIb/IIIa receptors), and CY-Chrome™ conjugated CD62P(recognizes p-selectin). All three antibodies were purchased from BDBiosciences, San Diego, Calif. The blood-citrate mixture was stimulatedwith 5 μM ADP for 2 minutes. Saturating concentrations of respectiveantibodies were then added and the tubes were incubated at roomtemperature for 20 minutes in the dark. The labeled samples were fixedby the addition of 1% buffered paraformaldehyde and stored at 4° C. forat least 2 hours. The labeled samples were analyzed by a BectonDickinson FACScan flow cytometer set up to measure fluorescence lightscatter. The instrument was calibrated with fluorescence beads for thethree-color flow cytometer setup (CaliBRITE™ 3, BD Biosciences). Aftersetting the gate around platelets, FL1 (FITC)/FL2 (R-PE) and FL2(R-PE)/FL3 (CY-Chrome™) compensations were adjusted. All the variableswere collected by use of 4-decade logarithmic amplification. The datawere collected in list mode and then analyzed using CELL Quest Software(BD Biosciences). P-selectin was expressed as percent positive cells(i.e. the ratio of CD62P (CY-Chrome™) verses CD41a (R-PE) positivecells) as previously described. Activated GP IIb/IIIa was expressed aslog mean fluorescence intensity.

Myocardial Necrosis Markers.

Cardiac markers were measured at the same times as the platelet assays.The peak levels of troponin I, creatinine kinase MB (CKMB), andmyoglobin were determined using the Triage® Cardiac Panel with a Triage®Meter (Biosite Inc., San Diego, Calif.). This method is based on afluorescence immunoassay for the quantitative determination of thesecardiac markers. The upper limit of normal (ULN) value for troponin I is1.0 ng/ml; for myoglobin is 107 ng/ml; and for CKMB is 4.3 ng/ml.

Definitions

Relative platelet inhibition was defined for purposes of this study asfollows: (baseline aggregation minus posttreatment aggregation)/baselineaggregation×100%.₁₄ Mean platelet reactivity was calculated as theaverage platelet aggregation recorded at 3, 8 and 18-24 hourspoststenting. Relative inhibition of active GP IIb/IIIa expression wasdefined as follows: (baseline MFI minus post-treatment MFI/baselineMFI). The definition of an infarct was CK-MB>3× upper limits normal(ULN) in at least 2 samples and a large infarct was defined asCK-MB>5×ULN in at least 2 samples._(10,13) Bleeding was quantifiedaccording to the TIMI criteria.₁₆ In brief, major bleeding was definedas clinically overt bleeding accompanied by a fall in hemoglobin of3.0-5.0 g/dl or a fall in hematocrit of 9 to <15%. Major bleedingoccurred when the hemoglobin decreased >5 g/dl or the hematocrit ≥15%.

Sample Size and Statistical Analysis.

Previous studies had shown that a 300 mg clopidogrel loading doseproduces <40% inhibition of baseline aggregation in response to 5 and 20μM ADP at 24 hours after administration. Other studies demonstrated >80%inhibition by eptifibatide. Using the statistical calculationm=2×[Z_((1-á/2))+Z_((1-â))]₂/_(0.2), where m=number of patients,statistical significance level (α)=5%, power (β)=90% and Δ=standardizeddifference; approximately 30 patients will be needed in each arm in thestudy. Comparisons were made between groups by one way analysis ofvariance (Statistica software, Tulsa, Okla.). The Wilks-Shapiro test wasused to assess conformity with a normal distribution. Based on thenormal distribution of data the mean±SEM is reported except as otherwisenoted and p<0.05 was considered significant.

Results

Patients.

One hundred and twenty patients were enrolled and had platelet assaysperformed. The clinical and angiographic demographics of the fourtreatment groups are shown in Tables 4 and 5, respectively.

TABLE 4 Patient Demographics Group A Group B Group C Group D (n = 30) (n= 30) (n = 30) (n = 30) Age (years) 68 ± 21  55 ± 30  58 ± 12 64 ± 9Race (Caucasian) n, 20 (67) 26 (87) 25 (84) 19 (64) (%) Gender (Male) n,(%) 13 (43) 19 (63) 22 (73) 18 (60) Risk Factors/Past medical Hx n, (%)Smoking 20 (67) 19 (64) 14 (46) 21 (70) Family history of CAD 16 (53) 18(60)  9 (30) 21 (70) Hypertension 21 (70) 20 (67)  20 (67)) 27 (90)Hyperlipidemia 21 (70) 24 (77) 26 (87) 26 (87) Diabetes 16 (53)  9 (30)12 (40) 11 (37) Prior Myocardial  7 (23) 10 (33)  8 (27)  8 (27)Infarction Prior CABG  5 (17)  5 (17)  6 (20) 3 (9) Prior PTCA 13 (43) 9 (30) 13 (43)  8 (27) Pretreatment Medications n, (%) Beta blockers 27(90) 25 (84) 27 (90) 29 (97) ACE Inhibitors 20 (67) 10 (64) 23 (77) 24(80) Calcium blockers  5 (17)  7 (23)  3 (9)  5 (17) Lipid loweringagents 3A4 Pathway 16 (53) 16 (53) 21 (70) 11 (37) metabolized Non 3A4Pathway  7 (24)  6 (20)  6 (20) 10 (33) metabolized Laboratory Data WBC(×1000/mm³) 8.2 ± 3.8  8.3 ± 3.6  7.9 ± 2.5  7.2 ± 1.8 Platelets(×1000/mm³) 250 ± 106 230 ± 90 232 ± 71 204 ± 34 Hemoglobin (g/dl) 12.4± 2.0  12.9 ± 1.8 13.7 ± 1.7 12.7 ± 2.0 Creatinine (g/dl) 1.2 ± 1.1 1.15± 0.4 0.89 ± 0.2  1.0 ± 0.2 Data Reported as Mean ± SD ACE = angiotensinconverting enzyme; CABG = coronary artery bypass graft surgery; CAD =coronary artery disease; PTCA = percutaneous coronary angioplasty; WBC =white blood cells

TABLE 5 Procedural Characteristics Group A Group B Group C Group D (n =30) (n = 30) (n = 30) (n = 30) Length of 72 ± 38 62 ± 19 67 ± 37 54 ± 17procedure (min.) Ejection 53 ± 8  55 ± 8  49 ± 11 53 ± 9  Fraction (%)Number of 1.3 ± 0.5 1.3 ± 0.6 1.4 ± 0.6 1.5 ± 0.7 vessels treated LesionMorphology Denovo n, (%) 29 (97) 26 (87) 26 (87)  27 (90) LesionLocation n, (%) LAD 10 (33)  9 (30) 13 (43)   9 (30) CX  4 (13)  9 (30)9 (30)  8 (27) RCA 14 (47) 11 (37) 5 (17) 12 (40) SVG 2 (7) 1 (3) 3 (10)1 (3) Stent Types n, (%) Drug eluting 16 (53) 22 (73) 18 (60)  20 (67)Bare metal 12 (40)  6 (20) 9 (30)  9 (30) PTCA only 2 (7) 2 (7) 3 (10) 1(3) Reference vessel 2.9 ± 0.5 3.2 ± 0.5  3 ± 0.4  3 ± 0.4 diameter (mm)Total lesion 18.5 ± 10  22.5 ± 15  22 ± 15 20 ± 12 length (mm)Pre-stenosis (%) 81 ± 8  85 ± 6  88 ± 5  83 ± 7  Post-stenosis (%)  2 ±0.5 4 ± 2 5 ± 3 4 ± 2 Procedural 30 (100) 28 (93) 29 (97)  28 (93)success n, (%) Data Reported as Mean ± SD CX = circumflex artery; LAD =left anterior descending artery; RCA = right coronary artery; SVG =saphenous vein graft

Group A was the oldest and Group C had the highest percentage of males.Cardiovascular risk factors were common and the incidence of diabeteswas high in all groups. One patient in each group had presented with anon-ST elevation myocardial infarction. The use of statins metabolizedby the CYP 3A4 pathway was the lowest in Group D. Concomitantmedications were frequently used in all groups. Multivesselinterventions were commonly performed and drug-eluting stents were oftenused. There were no in-hospital deaths. There was one ST-elevationmyocardial infarction that occurred in-hospital following a subacutethrombosis in a patient assigned to Group A.

There were no strokes or episodes of congestive heart failure. Hematomaswere the cause of all bleeding episodes. Minor bleeding occurred in onepatient in Group A and major bleeding occurred in 1 patient each inGroups C and D.

Platelet Aggregation.

FIG. 4 shows the pharmacodynamic responses in the four groups inresponse to 5 μM ADP. In the groups not treated with eptifibatide,baseline aggregation was 63±11% in Group A and 66±7% in Group B (p=NS).In the groups treated with eptifibatide, baseline aggregation was 58±11%in Group C and 62±7% in Group D (p=NS). In the groups not treated witheptifibatide, a 600 mg clopidogrel loading dose provided greaterplatelet inhibition throughout the first 24 hours after stenting. GroupB had greater inhibition than Group A at 3 hours (p<0.001); 8 hours(p<0.001); and 18-24 hours (p<0.001). The peak inhibitory effectfollowing a 600 mg loading dose occurred at 8 hours as compared to 18-24hours following a 300 mg loading dose. Groups C and D exhibited the sameinhibition (p=NS at all times) and both groups exhibited twofold greaterinhibition as compared to Groups A and B at all times (p<0.001).

FIG. 5 shows the platelet response to 20 μM ADP. Baseline aggregationdid not differ between groups. The groups receiving clopidogrel andeptifibatide had consistently the lowest reactivity over 24 hours(p<0.001 vs. Groups A and B at all times). At a 20 μM agonistconcentration Group D showed greater inhibition at 18-24 hours ascompared to Group C (p=0.05). Group B had the same inhibition as Group Aat 3 hours (p=0.55); and greater inhibition at 8 hours (p=0.09); and18-24 hours (p=0.01). The peak inhibitory effect following a 300 or 600mg loading dose was reached at 8 hours.

Flow Cytometry.

Stimulated p-selectin expression at baseline did not differ betweengroups. Post-treatment p-selectin expression was significantly reducedin all groups as compared to baseline expression whereas treatment with300 mg clopidogrel alone had the least effect in p-selectin expression(FIG. 6). Stimulated expression of active GP IIb/IIIa measured at 18-24hours was inhibited the most in Groups C (76±13%) and D (77±5%) ascompared to Groups A (50±20%, p<0.05) and B (63±7%, p<0.05) (FIG. 7).

Myocardial Necrosis Markers.

Overall, CKMB release (>1-3×ULN) was lowest in the groups treated witheptifibatide (p<0.005) (FIG. 8 and Table 6).

TABLE 6 Necrosis Markers Group A Group B Group C Group D Markers (n =30) (n = 30) (n = 30) (n = 30) CKMB (>1-3X ULN) (n) 6 6 2 1 CKMB (>3XULN) (n) 3 1 0 0 TN-I (>ULN) (n) 7 4 2 1 Myoglobin (>2X ULN) 7 6 3 0 (n)TN = troponin

Criteria were met for a myocardial infarction in 3 patients from Group Aand 1 from group B (p<0.03 for eptifibatide vs. clopidogrel alone).There were no large infarcts in either group that received eptifibatidewhereas 2 occurred in Group A and 1 in Group B. Similar findings wereobserved when troponin and myoglobin were measured (FIG. 9). A trend toless troponin elevation was observed in Group B as compared to Group A(p=0.10). Inhibition of either troponin I or myoglobin release was lowerin patients treated with eptifibatide+clopidogrel compared toclopidogrel alone (p=0.004 and p=0.002, respectively) (FIG. 9).

Relation of Platelet Reactivity to Myocardial Necrosis.

FIG. 10 demonstrates that eptifibatide use was associated with thelowest mean platelet reactivity and 300 mg clopidogrel alone wasassociated with higher mean platelet reactivity than 600 mg clopidogrelalone. FIG. 11 demonstrates the overall relation of myocardial necrosismarker release to mean platelet reactivity. Mean platelet reactivity wassignificantly higher in those patients who developed a myocardialinfarction or any increased myocardial marker release. Myocardialinfarction only occurred in those patients with mean 5 μM ADP-inducedaggregation greater than about 50% (FIG. 12). FIG. 12 demonstrates thatmean 20 μM ADP-induced aggregation was also correlated with myocardialinfarction.

The current study demonstrates that the incremental increases in cardiacmarker release observed in patients with higher levels of post-treatmentplatelet reactivity lend strong support to the concept that reactiveplatelets play a central role in the mediation of post-stent myocardialnecrosis. The absence of infarcts in patients with lower than about 50%mean 5 μM ADP-induced aggregation indicates a threshold effect. Thus a 5μM ADP-induced platelet aggregation score of less than 50%, regardlessof the percent reduction relative to pretreatment platelet aggregation,is a therapeutic target to which therapy should be tailored.

Superior early and consistent inhibition was observed over 24 hoursfollowing the high loading dose. For this reason, all pharmacodynamicstudies examining clopidogrel loading strategies in stenting shouldinclude serial analyses over at least an 18-24 hour time period. Therehave been no previous pharmacodynamic investigations examining theeffects of high clopidogrel loading doses with eptifibatide. This studysuggests that 600 mg clopidogrel may also add to the antiplatelet effectof eptifibatide at 18-24 hours, as supported by observation of loweraggregation following stimulation with a high concentration of agonist.The definition of large infarcts in the present study has correlatedwith mortality in previous investigations (Saucedo et al. J Am CollCardiol. 2000; 35:1134-41; Ellis et al. Circulation. 2002; 106:1205-10;Brener et al. J Am Coll Cardiol. 2002; 40:1961-7).

Example 3: Platelet Reactivity and Clot Strength are Risk Factors forthe Development of Ischemic Events within 6 Months

This example examines the association of platelet reactivity and rapidthrombin generation upon the incidence of long-term ischemic eventsfollowing coronary stenting. The following methods and materials wereused in this example.

Patients.

This study was approved by the Investigational Review Board at SinaiHospital of Baltimore. Consecutive patients undergoing non-emergentcoronary stenting provided informed consent prior to the procedure. Inorder to be included in the study patients had to undergo successfulpercutaneous revascularization and be discharged from the hospital.Patients who gave informed consent but received coronary bypass surgeryfor revascularization were excluded. All patients were over 18 yearsold. Other exclusion criteria were: a history of bleeding diathesis,acute myocardial infarction within 48 hours, elevated cardiac markers(above upper limits normal for the respective assay), cerebrovascularevent within 3 months, chronic vessel occlusion or angiographicallyvisible thrombus, illicit drug or alcohol abuse, prothrombin timegreater than 1.5 times control, platelet count <100,000/mm³, hematocrit<30%, creatinine >4.0 mg/dl, and glycoprotein (GP) IIb/IIIa use prior tothe procedure.

One hundred thirty-five patients received a loading dose of clopidogrel[300 mg (n=75), 600 mg (n=60)] in the catheterization laboratoryimmediately after successful stenting. Patients on a maintenance dose ofclopidogrel at the time of admission (n=57) did not receive a loadingdose. A GP IIb/IIIa inhibitor (n=92, all patients received eptifibatide)was administered at the discretion of the treating physician.Unfractionated heparin was administered according to the ESPRIT dosingregimen (60 U/kg) as a bolus to all patients receiving GP IIb/IIIainhibitors and was dosed to achieve an activated clotting time of 300seconds in those not treated with GP IIb/IIIa inhibitors (REF). Allpatients had received at least 81 mg aspirin for 7 days prior to theprocedure and 325 mg was administered on the day of the procedure anddaily thereafter. The maintenance dose of clopidogrel was 75 mg daily.

Blood Sampling.

Pretreatment blood samples were obtained in the catheterizationlaboratory before GP IIb/IIIa inhibitors or heparin administrationthrough the indwelling femoral vessel sheath and transferred tovacutainer blood collecting tubes (Becton-Dickinson, Franklin Lakes,N.J.) containing 3.8% trisodium citrate (for LTA Assay) or 40 USPlithium heparin (for thromboelastography platelet mapping assay) afterdiscarding the first 2-3 ml of free flowing blood. The vacutainer tubewas filled to capacity and gently inverted 3 to 5 times to ensurecomplete mixing of the anticoagulant. In those patients treated with GPIIb/IIIa inhibitors discharge blood samples were obtained at least 18hours after cessation of therapy. In the remaining patients thedischarge blood samples were obtained 24 hours post-procedure.

Light Transmittance Aggregometry (LTA).

Platelet aggregation was assessed as described previously (Matetzky etal. Circulation. 2004; 109:3171-5). Briefly, the blood-citrate tubeswere centrifuged at 120 g for 5 minutes to recover platelet rich plasma(PRP) and further centrifuged at 850 g for 10 minutes to recoverplatelet poor plasma (PPP). The PRP and PPP were stored at roomtemperature to be used within two hours. Platelets were stimulated with20 μM ADP and the aggregation was assessed using a ChronologLumi-Aggregometer (Model 490-4D) with the aggrolink software package(Chronolog, Havertown, Pa.). Aggregation was expressed as the maximumpercent change in light transmittance from baseline, using PPP as areference.

Thrombelastography Assay—Clot Strength, Thrombin Generation Time and ADPInduced Aggregation.

The TEG Hemostasis Analyzer 5000 Series with automated analyticalsoftware provides quantitative and qualitative measurements of thephysical properties of a clot (Samara et al. Thromb Res. 2005;115(1-2):89-94). In the present study, the maximum amplitude (MA) andthe reaction time (R) were measured for the thrombin generated clotsample. MA is an indicator of the viscoelasticity of clot formation orclot strength and is dependent on platelet aggregation and fibrinformation and polymerization. R is the period of time of latency untilinitial fibrin formation and has been correlated with the velocity ofthrombin generation (Rivard et al. Evaluation of the profile of thrombingeneration during the process of whole blood clotting as assessed bythromboelastography. (AHA 2004, Abstract)).

One mL heparinized blood was transferred to a vial containing kaolin andmixed by inversion. Five hundred microliters of the activated blood wasthen transferred to a vial containing heparinase and mixed to neutralizeheparin. The neutralized blood (360 μL) was added to a heparinase coatedcup and assayed in the thromboelastography according to themanufacturer's instructions to obtain the thrombin-induced clot(MA_(KH)). The thromboelastography analytical software generated the MAas well as R values.

Heparinized blood (360 μL) was added to a non-coated cup containingreptilase and factor XIIIa activator to generate a whole bloodcrosslinked clot in the absence of thrombin generation (MA₀). A thirdsample (360 μL) of heparinized blood was added to a plain cup in thepresence of the reptilase/XIIIa activator and ADP (2 μM) to generatewhole blood-crosslinked clot with platelet activation (MA_(ADP)).Platelet inhibition in response to ADP was calculated using computerizedsoftware {according to manufacturer's instructions based on the formula:{% Inhibition=[(MAADP−MA0)/(MAKH−MA0)]×100.} and Percent aggregation (%MAADP) was determined by subtracting the % MAADP value from 100.

Clinical Outcomes.

Patients were contacted by telephone at the end of 1 month and sixmonths to determine the occurrence of adverse events. Ischemic eventswere defined as the occurrence of death secondary to cardiovascularcause, myocardial infarction, stroke, stent thrombosis and recurrentischemia diagnosed by the treating physician that requiredrehospitalization. Patients were divided into 2 groups based on theoccurrence of adverse ischemic events.

Statistical Analysis.

Comparisons were made between the ischemic and non-ischemic groups byone way analysis of variance (Statistica software, Tulsa, Okla.). Basedon the normal distribution of data the mean±SD is reported except asotherwise noted and p<0.05 was considered significant. A multivariateanalysis was performed to determine risk factors for ischemic events.

Results Patients and Clinical Outcomes

One hundred and ninety-two patients underwent catheter-based treatmentand were analyzed. All of the procedures performed were non-emergent.Thirty six patients were admitted with unstable angina, and 11 patientshad non-ST elevation myocardial infarction. The remainder of thepatients had stable angina. The demographics and angiographic data ofthe patients with and without ischemic events are shown in Tables 7 and8, respectively.

TABLE 7 Patient Demographics Patients with Patients without IschemicEvents Ischemic Events p - (n = 38) (n = 154) Value Age (years)  59 ± 10 62 ± 12 NS Race (Caucasian) n, (%) 68 57 NS Gender (Male) n, (%) 42 600.05 BMI 31 ± 7 30 ± 7 NS Risk Factors/Past medical Hx (%) Smoking 39 45NS Family history 47 32 NS of CAD Hypertension 81 63 0.04 Hyperlipidemia92 57  0.001 Diabetes 50 40 NS Prior Myocardial 24 40 NS InfarctionPrior CABG 18 26 NS Prior PTCA Pretreatment Medications (%) Betablockers 90 81 NS ACE Inhibitors 74 61 NS Calcium blockers 21 22 NSLipid lowering agents 3A4 Pathway 74 61 NS metabolized Non 3A4 Pathway18 24 NS metabolized Laboratory Data WBC (×1000/mm³)  7.3 ± 2.3  7.6 ±2.4 NS Platelets (×1000/mm³) 244 ± 79 222 ± 66 NS Hemoglobin (g/dl) 12.7± 2.3 13.3 ± 1.8 NS Creatinine (g/dl)  1.1 ± 0.6  1.1 ± 1.8 NS DataReported as Mean ± SD; ACE = angiotensin converting enzyme; BMI = bodymass index; CABG = coronary artery bypass graft surgery; CAD = coronaryartery disease; PTCA = percutaneous coronary angioplasty; WBC = whiteblood cells; 3A4 = hepatic cytochrome 3A4

TABLE 8 Procedural Characteristics Patients With Patients WithoutIschemic Events Ischemic Events p - (n = 38) (n = 154) Value Length of55.4 ± 22.3 62.1 ± 33.5 NS procedure (min.) Ejection 47.5 ± 8.5  51.5 ±9.0  NS Fraction (%) Number of 1.25 ± 0.5  1.32 ± 0.6  NS vesselstreated Lesion Morphology Denovo (%) 87 89 NS Culprit Lesion Location(%) LAD 40 38 NS CX 21 25 NS RCA 34 30 NS SVG 5 7 NS Stent Types (%)Drug eluting 75 68 NS Bare metal 18 29 NS PTCA only 7 3 NS Referencevessel 3.0 ± 0.4 3.0 ± 0.5 NS diameter (mm) Total lesion 21.9 ± 10.119.0 ± 12.2 NS length (mm) Pre-stenosis (%) 86 84 NS Post-stenosis (%) 55 NS Procedural 95 96 NS success (%) Data Reported as Mean ± SD; CX =circumflex artery; LAD = left anterior descending artery; RCA = rightcoronary artery; SVG = saphenous vein graft

There were more male patients without ischemia. Hypertension andhyperlipidemia were more common in patients with ischemia. There were nodifferences in medications between the two groups. Multivesselinterventions were commonly performed and drug-eluting stents were oftenused. There were 4 in-hospital ischemic events. All of these patientshad myocardial infarction. One of these patients had stent thrombosis.

Six month follow-up data were complete in 191/192 patients. There were44 events that occurred in 38 patients (20%) within 6 months ofdischarge (FIG. 13). All events occurred during aspirin therapy.Thirty-two patients were receiving dual antiplatelet therapy at the timeof their first event (about 17% treatment failure rate). Six patientshad 2 events.

At one month post-discharge 20 of 191 patients (about 10%) had events.These events were: myocardial infarction (n=2), ischemia requiringrevascularization of the prior target vessel (TVR) (n=2 patients),ischemia involving a vessel other than the prior target vessel requiringrevascularization (non-TVR) (n=6), ischemia requiring hospitalizationbut not revascularization (n=9), and stroke (n=1). At 1-6 months 18patients had the first occurrence of an event. These events were: death(n=2), ischemia requiring TVR (n=6), ischemia requiring non-TVR (n=4),ischemia requiring hospitalization but not revascularization (n=6). Sixpatients had the occurrence of a second event at 1-6 months. Theseevents were: coronary artery bypass grafting (n=2), ischemia requiringTVR (n=1), ischemia requiring non-TVR (n=1), and ischemia requiringhospitalization but not revascularization (n=2)

Platelet Function Studies

One hundred-sixty patients had platelet aggregation measured by LTA and192 samples were analyzed by thromboelastography (Table 9). Pretreatmentaggregation by LTA was 73±9% in patients with ischemic events and 74±10in patients without ischemic events (p=NS). There was no significantdifference in platelet aggregation measured by thromboelastography(p=NS).

TABLE 9 Evaluation of Platelet Function Tests by Light TransmittanceAggregometry and Thrombelastography Patients With Patients Without p-Ischemic Events Ischemic Events Value 20 μM ADP-Induced 73 ± 9  74 ± 10NS Pre-treatment Aggregation (%) 20 μM ADP-Induced 63 ± 12 56 ± 16 0.02Post-treatment Aggregation (%) THROMBO- 76 ± 14 71 ± 20 NS ELASTOGRAPHY% MA_(ADP) Pre-treatment THROMBO- 59.3 ± 19  50 ± 20 0.02 ELASTOGRAPHY %MA_(ADP) Post-treatment THROMBO- 73.9 ± 4.8  64.5 ± 4.0  <0.001ELASTOGRAPHY MA (mm) Reaction Time 4.3 ± 1.3 4.6 ± 2.0 NS Pre-treatment(min) Reaction Time 4.3 ± 1.3 5.9 ± 1.5 <0.001 Post-treatment(min)

Patients with ischemic events demonstrated a higher mean dischargeplatelet aggregation by LTA than patients without ischemic events(p=0.02, Table 9, FIGS. 14 and 15). Similarly, discharge plateletaggregation measured by thromboelstography was higher in patients withevents than in patients without ischemic events (Table 9, p=0.02).Thomboelastography MA and thromboelastography R was also assessed inpatient with and without ischemic events (FIGS. 18 and 19) The greatestfrequency of patients with ischemic events was present in the highestquartiles of platelet reactivity measured by either LTA orthromboelastography measurement of maximum clot strength (MA) (FIGS. 16and 17, respectively). Patients with ischemic events had significantlygreater clot strength than patients without ischemic events p=<0.001)(FIG. 16). The reaction time R, was significantly shorter in patientswith events (p=<0.001) (FIG. 18). Patients in the lowest two quartilesreaction time had the highest incidence of ischemic events (FIG. 19).

This example demonstrates that high ex-vivo platelet reactivity to ADPand (as well as) clot strength, and rapid thrombin generation measuredat discharge are independent risk factors for the development ofischemic events within 6 months of elective coronary artery stenting.Since clot strength following stimulation with kaolin as measured bythromboelastography is dependent on platelet aggregation by thrombin,our findings strongly support the central role of platelet reactivity toboth thrombin and ADP in atherothrombosis. All of these data indicatethat the risk of in vivo platelet-related events can be assessed, inpart, by ex vivo testing.

This example further demonstrates a near absence of ischemic events inpatients with the lowest quartile of aggregation measured by LTA afterADP stimulation. However, since approximately 50% of events occurred inpatients with average reactivity to ADP, an agonist other than ADP maybe playing a dominant role in the genesis of ischemia. Moreover, 78% ofpatients suffering events had the highest clot strength measured bythromboelastography. Therefore maximum responsiveness to thrombin alsoappears to predict outcomes. Thus, in those patients with averageresponsiveness to ADP, high reactivity to thrombin may be of centralimportance in ischemic event occurrence. This may be an explanation forischemic events occurring in patients despite inhibition of ADP-inducedaggregation by clopidogrel. Finally, these data indicate that thevelocity of thrombin generation is also an independent risk factor thatmay be particularly relevant in patients with vigorous plateletaggregation in response to thrombin.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1.-45. (canceled)
 46. A method of treating a vascular disease in apatient receiving an anti-platelet inhibitor therapy, the methodcomprising: administering an adjusted platelet inhibitor therapy to thepatient so as to reduce platelet reactivity, or to increase at least oneof time-to-thrombin formation (TTF), or time-to-fibrin formation (TFF),when a test score for the patient is greater than a risk threshold scorebased on a population study and thus indicating the patient is at riskof a thrombotic event, wherein the test score is determined by assayingin vitro platelet function in a blood sample from the patient, whereinplatelet function is assayed by assaying platelet reactivity, TTF, orTFF in the blood sample in response to an agonist, and wherein assayingplatelet reactivity is by a method other than assaying GP IIb/IIIareceptor expression, said assaying platelet function providing the testscore, and wherein the test score is independent of a pre-treatmentbaseline of platelet reactivity in the patient.
 47. The method of claim46, wherein the thrombotic event is myocardial ischemia.
 48. The methodof claim 46, wherein the thrombotic event is a thrombosis.
 49. Themethod of claim 48, wherein the patient has a stent and the thrombosisis a stent thrombosis.
 50. The method of claim 46, wherein thethrombotic event is myocardial infarction, unstable angina, stableangina, restenosis, stroke or deep vein thrombosis.
 51. The method ofclaim 46, wherein the risk of a thrombotic event is risk of a thromboticevent within about 4 months to 12 months.
 52. The method of claim 46,wherein the risk of a thrombotic event is risk of a thrombotic eventwithin about 6 months to 12 months.
 53. The method of claim 46, whereinthe risk of a thrombotic event is risk of a thrombotic event withinabout 18 months.
 54. The method of claim 46, wherein the risk of athrombotic event is risk of a thrombotic event within about 6 months.55. The method of claim 46, wherein the platelet inhibitor therapycomprises an anti-thrombotic agent selected from the group consisting ofclopidogrel, enoxaparin, hirudin, hirulog and argatroban.
 56. The methodof claim 46, wherein TTF or TFF is assayed.
 57. The method of claim 56,wherein TTF and TFF are assayed by thromboelastography R and the riskthreshold score is from about 4.6 min to 5.6 min.
 58. The method ofclaim 46, wherein platelet reactivity is assayed by a method other thanthromboelastography maximum amplitude (MA).
 59. The method of claim 46,wherein the platelet reactivity is assayed by 5 μM ADP-induced plateletaggregation and the risk threshold score is from about 24% to 36%aggregation.
 60. The method of claim 59, wherein the platelet inhibitortherapy comprises an anti-thrombotic agent selected from the groupconsisting of clopidogrel, enoxaparin, hirudin, hirulog and argatroban.61. The method of claim 46, wherein the platelet reactivity is assayedby 20 μM ADP-induced platelet aggregation and the risk threshold scoreis from about 40% to 60% aggregation.
 62. The method of claim 61,wherein the platelet inhibitor therapy comprises an anti-thromboticagent selected from the group consisting of clopidogrel, enoxaparin,hirudin, hirulog and argatroban.
 63. The method of claim 46, wherein theplatelet reactivity is assayed by a P2Y₁₂ reactivity ratio and the riskthreshold score is from about 32 to about
 48. 64. The method of claim63, wherein the platelet inhibitor therapy comprises an anti-thromboticagent selected from the group consisting of clopidogrel, enoxaparin,hirudin, hirulog and argatroban.